Chondropsin-class antitumor v-atpase inhibitor compounds, compositions and methods of use thereof

ABSTRACT

A composition comprising a substantially purified compound of the formula: 
     
       
         
         
             
             
         
       
     
     in combination with at least one additional therapeutic agent, and methods of preventing or treating cancer and a condition treatable by the inhibition of vacuolar-type (H+)-ATPase.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 10/521,930, filed on Apr. 18, 2005, which is a U.S.national phase of International Patent Application No.PCT/US2003/023290, filed on Jul. 24, 2003, and which claims the benefitof U.S. Provisional Patent Application No. 60/398,092, which was filedon Jul. 24, 2002.

FIELD OF THE INVENTION

The present invention relates to vacuolar-type (H+)-ATPase-inhibitingmacrocyclic lactams belonging to the chondropsin class of compounds,compositions, and methods of using them.

BACKGROUND OF THE INVENTION

Vacuolar (or vacuolar-type or V-type) (H+)-ATPases have been describedas “a universal proton pump of eukaryotes” (Finbour and Harrison,Biochem. J., 324, 697-712 (1997)). Vacuolar-type (H+)-ATPases arepresent in many tissues and cells of the body. Intracellular vacuolar(H+)-ATPase activities are present in certain organelles, and areresponsible for maintaining the internal acidity thereof. Thismaintenance is essential for a variety of physiological functions suchas: sorting of membrane and organellar proteins; proinsulin conversion;neurotransmitter uptake; cellular degradative processes; and, receptorcycling. See Mellman et al., Ann. Rev. Biochem., 55, 663-699 (1986);Forgac, Physiological Rev., 69, 765-796 (1989); Stevens and Forgac,Annu. Rev. Cell. Dev. Biol., 13, 779-808 (1997); Nelson, TIPS, 12, 71-75(1991).

Vacuolar-type (H+)-ATPase activity is also located within specializedplasma membranes. Important examples include the vacuolar-type(H+)-ATPase activity in the plasma membranes of kidney intercalatedcells, osteoclasts and sperm cells. See Stone and Xie, Kidney Int., 33,767-774 (1988); Vaananen et al., J. Cell, Biol., 111, 1305-1311 (1990);Blair et al., Science, 245, 855-857 (1987); Wang and Gluck, J. Biol.Chem., 265, 21957-21965 (1990); Hall and Chambers, Inflamm. Res., 45,1-9 (1996); Hall and Schaueblin, Bone and Mineral, 27, 159-166 (1994);David and Baron, Exp. Opin. Invest. Drugs, 4, 725-740 (1995); Wassarman,Science, 235, 553-560 (1987); Nelson, TIPS, 12, 71-75 (1991).

Because of the importance of vacuolar-type (H+)-ATPase activity in themaintenance of many physiological functions, compounds which inhibitvacuolar-type (H+)-ATPase will have useful pharmacological applicationsin a variety of different situations. See reviews by Nelson, TIPS, 12,71-74 (1991), and Keeling et al., Ann. New York Acad. Sci., 834, 600-608(1997), and references contained therein. For example, a givenvacuolar-type (H+)-ATPase inhibitor may have utility against one or moredisease states or physiological functions, in which it is desirable toinhibit an intra-organellar, vacuolar-type (H+)-ATPase-mediated process,such as acidification, accumulation of a neurotransmitter, receptorturnover, lysosomal storage, and the like. See Mellman et al., Ann. Rev.Biochem., 55, 663-699 (1986); Forgac, Physiological Rev., 69, 765-796(1989); Stevens and Forgac, Annu. Rev. Cell. Dev. Biol., 13, 779-808(1997); Nelson, TIPS, 12, 71-75 (1991). Similarly, a given vacuolar-type(H+)-ATPase inhibitor may be useful against one or more disease statesor physiological functions, in which it is desirable to modify a plasmamembrane vacuolar-type (H+)-ATPase-mediated process, such as urinaryacidification, bone resorption, or the acrosomal acid secretion requiredfor fertility. See Stone and Xie, Kidney Int., 33, 767-774 (1988);Vaananen et al, J. Cell. Biol., 111, 1305-1311 (1990); Blair et al.,Science, 245, 855-857 (1987); Wang and Gluck, J. Biol. Chem., 265,21957-21965 (1990); Hall and Chambers, Inflamm. Res., 45, 1-9 (1996);Hall and Schaueblin, Bone and Mineral, 27, 159-166 (1994); David andBaron, Exp. Opin. Invest. Drugs, 4, 725-740 (1995); Wassarman, Science,235, 553-560 (1987); Nelson, TIPS, 12, 71-75, (1991).

Compounds that inhibit vacuolar-type (H+)-ATPases also will haveimportant utility for cancer therapy. For example, there is literatureevidence indicating involvement of vacuolar-type (H+)-ATPases inprocesses related to cellular proliferation, angiogenesis, tumor cellinvasiveness, metastasis, and drug resistance (see, e.g., Akifusa et.al., Exp. Cell Res., 238, 82-89 (1998); Altan et al., J. Exp. Med., 187,1583-1598 (1998); Gerard et al., J. Exp. Biol., 201, 21-31 (1998); Ishiiet al., J. Antibiot., 48, 12-20 (1995); Moriyama et al., J. Biochem.,115, 213-218 (1994); Ohkuma et al., In Vitro Cell Devel. Biol., 29A,862-866 (1993); Perona et al., Nature, 334, 438-440 (1988); Montcourrieret al., J. Cell Sci., 107, 2381-2391 (1994); Montcourrier et al., Clin.Exp. Metastatis, 15, 382-392 (1997); Martinez-Zaguilan et al., Ann. NYAcad. Sci., 671, 478-480 (1992); Martinez-Zaguilan et al., Am. J.Physiol., 265, C1015-C1029 (1993); Martinez-Zaguilan et al., J. Cell.Physiol., 176, 196-205 (1998); Nishihara et al., Biochem. Biophys. Res.Commun., 212, 255-262 (1995); Manabe et al., J. Cell Physiol., 157,445-452 (1993); Kinoshita et al., FEBS Lett., 337, 221-225 (1994);Kinoshita et al., FEBS Lett., 398, 61-66 (1996); Ohta et al., Brit. J.Cancer, 73, 1511-1517 (1996); Ohta et al., J. Pathol., 185, 324-330(1998); Marquardt et al., J. Natl. Cancer Inst., 83, 1098-1102 (1991);and Banderra et al., Int. J. Oncol., 12, 711-715 (1998)). Therefore,compounds that inhibit these phenomena will be useful in cancerchemotherapy.

Among the numerous challenges faced by medicinal chemistry research isthe challenge of identifying new vacuolar-type (H+)-ATPase-inhibitoryleads applicable to medical treatments. In addition, the identificationand development of new leads useful in cancer chemotherapy remains aperplexing problem. Purely synthetic approaches toward theidentification of novel anticancer agents and vacuolar-type (H+)-ATPaseinhibiting agents have been typically unsuccessful, partly due to thetechnological and human limitations inherent in laboratory synthesis.Although biological metabolites provide a vast resource of newstructurally diverse chemical compounds, the number of agents availablefor exploiting therapeutic opportunities are relatively few,particularly inhibitors of vacuolar-type (H+)-ATPase. For example,structural types that potently and selectively inhibit vacuolar-type(H+)-ATPases have thus far been limited to compounds such asbafilomycins, concanamycins, and benzolactone enamides such as thesalicylihalamides and lobatamides (see Boyd, PCT International PatentApplication No. PCT/US00/05582).

Thus, there remains a need for new vacuolar-type (H+)-ATPase inhibitorsand anticancer compounds, pharmaceutical compositions, and methods ofusing them. The present invention provides such compounds, compositions,and methods. These and other advantages of the present invention, aswell as additional inventive features, will be apparent from thedescription of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a substantially purified compound of theformula:

wherein:

R¹ is H, a straight-chain or branched C₁₋₃₀ saturated alkyl, astraight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹ isunsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(1a), CO₂R^(1a), and OC(O)R^(1a), wherein R^(1a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof;

R²-R⁸ are the same or different and each is R¹⁰, C(O)R¹⁰, SO₃R¹⁰, orSO₂R¹⁰, wherein R¹⁰ is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹⁰is unsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(10a), CO₂R^(10a) and OC(O)R^(10a), wherein R^(10a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; and

R⁹ is a substituent of the formula:

wherein the R^(9a) substituents are the same or different and each isR¹¹, C(O)R¹¹, or SO₂R¹¹, wherein R¹¹ is H, a straight-chain or branchedC₁₋₃₀ saturated alkyl, a straight-chain or branched C₂₋₃₀ unsaturatedalkyl, or an aryl comprising 6-10 carbon atoms in the ring skeletonthereof, wherein R¹¹ is unsubstituted or substituted with one or moresubstituents, which are the same or different, selected from the groupconsisting of a halogen, an oxo, OR^(11a), CO₂R^(11a) and OC(O)R^(11a),wherein R^(11a) is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof;

wherein R^(1a), R^(10a) and R^(11a) are unsubstituted or substitutedwith one or more substituents selected from the group consisting of ahalogen, an oxo, and a hydroxyl. The compound of the present inventioncan be in the form of a pharmaceutically acceptable salt or a prodrug.

The present invention also provides a compound of the formula:

wherein:

R¹ is H, a straight-chain or branched C₁₋₃₀ saturated alkyl, astraight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹ isunsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(1a), CO₂R^(1a), and OC(O)R^(1a), wherein R^(1a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof;

R²-R⁸ are the same or different and each is R¹⁰, C(O)R¹⁰, SO₃R¹⁰, orSO₂R¹⁰, wherein R¹⁰ is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹⁰is unsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(10a), CO₂R^(10a) and OC(O)R^(10a), wherein R^(10a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; and

R⁹ is a substituent of the formula:

wherein the R^(9a) substituents are the same or different and each isR¹¹, C(O)R¹¹, or SO₂R¹¹, wherein R¹¹ is H, a straight-chain or branchedC₁₋₃₀ saturated alkyl, a straight-chain or branched C₂₋₃₀ unsaturatedalkyl, or an aryl comprising 6-10 carbon atoms in the ring skeletonthereof, wherein R¹¹ is unsubstituted or substituted with one or moresubstituents, which are the same or different, selected from the groupconsisting of a halogen, an oxo, OR^(11a), CO₂R^(11a) and OC(O)R^(11a),wherein R^(11a) is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof;

wherein R^(1a), R^(10a) and R^(11a) are unsubstituted or substitutedwith one or more substituents selected from the group consisting of ahalogen, an oxo, and a hydroxyl;

or a pharmaceutically acceptable salt thereof, provided that thecompound is not poecillastrin A.

The present invention additionally provides a composition comprising atherapeutically effective amount of at least one compound of the presentinvention, alone or in combination with at least one additionaltherapeutic agent. The therapeutically effect amount can be avacuolar-type (H+)-ATPase-inhibiting effective amount and/or ananticancer effective amount.

The present invention further provides a method of preventing ortreating a patient for a condition treatable by the inhibition ofvacuolar-type (H+)-ATPase, said method comprises administering to thepatient a vacuolar-type (H+)-ATPase-inhibiting effective amount of atleast one compound of the present invention, whereupon the patient istreated for the condition.

The present invention also further provides a method of preventing ortreating a patient for cancer, which method comprises administering tothe patient an anticancer effective amount of at least one compound ofthe present invention, whereupon the patient is treated for cancer.

The compound(s) used in accordance with the present invention can beadministered alone or in combination with a therapeutically effectiveamount of at least one additional therapeutic agent other than acompound of the present invention. Additional therapeutic agentsinclude, for example, vacuolar-type (H⁺)-ATPase inhibitors andanticancer compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the GI₅₀-based mean-graph “fingerprint” of anextract of Poecillastra species in the NCI 60 cell-line screen.

FIG. 1B illustrates the TGI-based mean-graph “fingerprint” of an extractof Poecillastra species in the NCI 60 cell-line screen.

FIG. 1C illustrates the LC₅₀-based mean-graph “fingerprint” of anextract of Poecillastra species in the NCI 60 cell-line screen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a substantially purified compound of theformula:

wherein:

R¹ is H, a straight-chain or branched C₁₋₃₀ saturated alkyl, astraight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹ isunsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(1a), CO₂R^(1a), and OC(O)R^(1a), wherein R^(1a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof;

R²-R⁸ are the same or different and each is R¹⁰, C(O)R¹⁰, SO₃R¹⁰, orSO₂R′°, wherein R¹⁰ is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹⁰is unsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(10a), CO₂R^(10a) and OC(O)R^(10a), wherein R^(10a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; and

R⁹ is a substituent of the formula:

wherein the R^(9a) substituents are the same or different and each isR¹¹, C(O)R¹¹, or SO₂R¹¹, wherein R¹¹ is H, a straight-chain or branchedC₁₋₃₀ saturated alkyl, a straight-chain or branched C₂₋₃₀ unsaturatedalkyl, or an aryl comprising 6-10 carbon atoms in the ring skeletonthereof, wherein R¹¹ is unsubstituted or substituted with one or moresubstituents, which are the same or different, selected from the groupconsisting of a halogen, an oxo, OR^(11a), CO₂R^(11a) and OC(O)R^(11a),wherein R^(11a) is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof;

wherein R^(1a), R^(10a) and R^(11a) are unsubstituted or substitutedwith one or more substituents selected from the group consisting of ahalogen, an oxo, and a hydroxyl. The compound of the present inventioncan be in the form of a pharmaceutically acceptable salt or a prodrug.

Preferred substituents for R¹-R⁸ include H and a straight-chain orbranched C₁₋₃₀ saturated alkyl. Substituent R³ is preferably hydrogen ormethyl. Preferred R^(9a) substituents include H and a straight-chain orbranched C₁₋₃₀ saturated alkyl. More preferably R^(9a) is H.

In a preferred embodiment, R³ is methyl and R¹, R², R⁴-R⁸ and R^(9a) areH.

Another embodiment of the invention includes a compound of the formula:

wherein:

R¹ is H, a straight-chain or branched C₁₋₃₀ saturated alkyl, astraight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹ isunsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(1a), CO₂R^(1a), and OC(O)R^(1a), wherein R^(1a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof;

R²-R⁸ are the same or different and each is R¹⁰, C(O)R¹⁰, SO₃R¹⁰, orSO₂R¹⁰, wherein R¹⁰ is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹⁰is unsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(10a), CO₂R^(10a) and OC(O)R^(10a), wherein R^(10a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; and

R⁹ is a substituent of the formula:

wherein the R^(9a) substituents are the same or different and each isR¹¹, C(O)R¹¹, or SO₂R¹¹, wherein R¹¹ is H, a straight-chain or branchedC₁₋₃₀ saturated alkyl, a straight-chain or branched C₂₋₃₀ unsaturatedalkyl, or an aryl comprising 6-10 carbon atoms in the ring skeletonthereof, wherein R¹¹ is unsubstituted or substituted with one or moresubstituents, which are the same or different, selected from the groupconsisting of a halogen, an oxo, OR^(11a), CO₂R^(11a) and OC(O)R^(11a),wherein R^(11a) is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof;

wherein R^(1a), R^(10a) and R^(11a) are unsubstituted or substitutedwith one or more substituents selected from the group consisting of ahalogen, an oxo, and a hydroxyl;

or a pharmaceutically acceptable salt thereof, provided that thecompound is not poecillastrin A.

In a particularly preferred embodiment, the present invention includes asubstantially purified compound of the formula:

Another particularly preferred embodiment includes a substantiallypurified compound of the formula:

The term “saturated alkyl” means a straight-chain or branched-chainsaturated alkyl which can contain from about 1 to about 30 carbon atoms,for example, from about 1 to about 20 carbon atoms, from 1 to about 10carbon atoms, from about 1 to about 8 carbon atoms, or from about 1 toabout 6 carbon atoms. Examples of saturated alkyls include methyl,ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,pentyl, isoamyl, hexyl, octyl, dodecanyl, octadecyl, and the like.Saturated alkyl substituents can be unsubstituted or substituted, forexample, with at least one substituent selected from the groupconsisting of a halogen, a nitro, an amino, a hydroxyl, a thio, an acyl,an alkyl, and a cyano.

The term “unsaturated alkyl” means saturated alkyl (straight-chain orbranched-chain), as defined herein, in which one or more of the singlecarbon-carbon bonds thereof is instead a multiple bond, for example, adouble or a triple bond. Thus, unsaturated alkyls include alkenyl andalkynyl substituents, as well as substituents that have a combination ofdouble and triple bonds. The term “alkenyl” means a straight-chain orbranched-chain alkenyl having one or more double bonds. Unless otherwisespecified, the alkenyl can contain from about 2 to about 30 carbonatoms, for example, from about 2 to about 20 carbon atoms, from about 2to about 10 carbon atoms, from about 2 to about 8 carbon atoms, or fromabout 2 to about 6 carbon atoms. Examples of alkenyls include vinyl,allyl, 1,4-butadienyl, isopropenyl, and the like. The term “alkynyl”means a straight-chain or branched-chain alkynyl radical having one ormore triple bonds. Unless otherwise specified, alkynyls can contain fromabout 2 to about 30 carbon atoms, for example, from about 2 to about 20carbon atoms, from about 2 to about 10 carbon atoms, from about 2 toabout 8 carbon atoms, or from about 2 to about 6 carbon atoms. Examplesof alkynyls include ethynyl, propynyl (propargyl), butynyl, and thelike. Unsaturated alkyl substituents can be unsubstituted orsubstituted, for example, with at least one substituent selected fromthe group consisting of a halogen, a nitro, an amino, a hydroxyl, athio, an acyl, an alkyl, and a cyano.

The term “aryl” means an aromatic carbocyclic radical, as commonlyunderstood in the art, and includes monocyclic and polycyclic aromaticssuch as, for example, phenyl and naphthyl rings. Preferably, the arylcomprises one or more six-membered rings including, for example, phenyl,naphthyl, biphenyl and the like. More preferably, the aryl comprisesabout six to about ten carbons. Aryl substituents can be unsubstitutedor substituted, for example, with at least one substituent selected fromthe group consisting of a halogen, a nitro, an amino, a hydroxyl, athio, an acyl, an alkyl, and a cyano.

It will be appreciated that the compounds of the present invention canbe obtained by methods known to those of skill in the art, for example,by structurally modifying poecillastrin A, or by direct synthesis, usingroutine synthetic transformations that are well known in the art. One ormore hydroxyl groups, for example, can be converted to the oxoderivative by direct oxidation. Direct oxidation can be accomplishedusing any known method such as, for example, a Swern oxidation, or byreaction with a metal oxidant such as a chromium oxide (e.g., chromiumtrioxide), a manganese oxide (e.g., manganese dioxide or permanganate)or the like. Primary alcohols can be oxidized to aldehydes, for example,via Swern oxidation, or they can be oxidized to carboxylic acids (e.g.,CO₂H), for example, by reaction with a metal oxidant as describedherein. Similarly, thiols (e.g., SR, SH or the like) can be converted tooxidized sulfur derivatives (e.g., SO₂R, SO₃H, or the like) by reactionwith an appropriate oxidant.

One or more hydroxyl groups also can be converted to an ester (e.g.,CO₂R) by reaction with an appropriate esterifying agent such as, forexample, an anhydride (e.g., (R(CO))₂O) or an acid chloride (e.g.,R(CO)Cl), or the like, or converted to a sulfonate (e.g., SO₂R) byreaction with an appropriate sulfonating agent such as, for example, asulfonyl chloride (e.g., RSO₂Cl), or the like, wherein R is any suitablesubstituent including, for example, organic substituents describedherein. Carboxylate esters also can be obtained by reacting one or morecarboxylic acids (e.g., CO₂H) with an alkylating agent such as, forexample, a diazoalkane (e.g., diazomethane), an alkyl or aryl iodide, orthe like. One or more amides can be obtained by reaction of one or morecarboxylic acids with an amine under appropriate amide-formingconditions. Appropriate amide-forming conditions include, for example,activation of a carboxylic acid (e.g., by conversion to an acid chlorideor by reaction with a carbodiimide reagent) followed by coupling of theactivated species with a suitable amine.

One or more hydroxyl groups also can be converted to a halogen atomusing a halogenating agent such as, for example, an N-halosuccinimidesuch as N-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide orthe like, in the presence of a suitable activating agent (e.g., aphosphine or the like). One or more hydroxyl groups also can beconverted to an ether by reacting one or more hydroxyls, for example,with an alkylating agent in the presence of a suitable base. Suitablealkylating agents can include, for example, an alkyl or aryl sulfonate,an alkyl or aryl halide, or the like. One or more suitably activatedhydroxyls, for example, a sulfonate ester, and/or one or more suitablyactive halides, can be converted to the corresponding thiol, cyano,halo, or amino derivative by displacement with a nucleophile. Suitablenucleophiles can include, for example, a thiol, a cyano, a halide ion,an amine (e.g., NH₂R⁹, wherein R⁹ is as described herein), or the like.

Functional groups such as, for example, amines can be obtained by avariety of methods known in the art. Amines can be obtained byhydrolysis of one or more amides such as, for example, one or more ofthe amides in poecillastrin A. Amines also can be obtained by reactingone or more suitable oxo groups (e.g., an aldehyde or a ketone) with oneor more suitable amines under the appropriate conditions, for example,reductive amination conditions, or the like. One or more amines, inturn, can be converted to a number of other useful derivatives such as,for example, amides, sulfonamides and the like.

Other structural modifications can be accomplished by incorporatingsynthetic, semisynthetic or naturally occurring materials such as, forexample, one or more amino acids, into the structure of one or morecompounds of formula (I). For example, modifications of R¹ and/or R⁸ canbe accomplished by incorporating different amino acids into themacrocyclic ring skeleton of formula (I). Such amino acids can include,for example, aspartic acid, phenyl alanine, serine, leucine, analogsthereof, homologs thereof, and the like. It will be appreciated that anumber of other synthetic transformations can be accomplished, otherthan those described herein, using routine chemistry that is well knownin the art. As such, the transformations and structural modificationsdescribed herein are in no way limiting, but are only illustrative forpreparing various compounds of the present invention.

Surprisingly and unexpectedly it has been found that compounds offormula (I) have anticancer activity and, further surprisingly,vacuolar-type (H+)-ATPase inhibitory activity. The compounds of thepresent invention can be obtained by one of ordinary skill in the art byisolation from natural sources; chemical synthesis using well-known andreadily available chemical reactions, reagents, and procedures; bysemisynthesis; or the like. The structure of formula (I) furthermoreprovides a practical template that can be used to produce a vast numberof structurally diverse, yet synthetically accessible, vacuolar-type(H+)-ATPase inhibitors and anticancer compounds.

One or more compounds of the present invention can be included in acomposition, e.g., a pharmaceutical composition. In that respect, thepresent invention further provides a composition that includes atherapeutically effective amount of at least one compound of the presentinvention and a pharmaceutically acceptable carrier. The therapeuticallyeffective amount can include an amount that produces a therapeutic orprophylactic response in a patient to whom a compound or composition ofthe present invention is administered. A therapeutically effectiveamount can include, for example, a vacuolar-type (H+)-ATPase-inhibitingeffective amount and/or an anticancer effective amount.

The composition of the present invention can further include atherapeutically effective amount of at least one additional compoundother than a compound of the present invention, for example, a compoundother than a compound of formula (I). When an additional compound isincluded in the composition of the present invention, the additionalcompound can be a vacuolar-type (H+)-ATPase-inhibiting compound (e.g., aconcanamycin or a bafilomycin or a benzolactone enamide, such as asalicylihalamide or a lobatamide). One or more additional anticancercompounds, other than a compound of the present invention, also can beincluded. When the additional compound is a vacuolar-type(H+)-ATPase-inhibitor other than a compound of the present invention, itis preferably present in the composition in a vacuolar-type(H+)-ATPase-inhibiting effective amount. When the additional compound isan anticancer compound, it is preferably present in the composition ofthe present invention in an anticancer effective amount.

The composition of the present invention can be produced by combiningone or more compounds of the present invention with an appropriatepharmaceutically acceptable carrier, and can be formulated into asuitable preparation. Suitable preparations include, for example,preparations in solid, semi-solid, liquid, or gaseous forms such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, and aerosols, and otherformulations known in the art for their respective routes ofadministration. In pharmaceutical dosage forms, a compound of thepresent invention can be used alone or in appropriate association, aswell as in combination, with other pharmaceutically active compounds,including other vacuolar-type (H+)-ATPase inhibiting compounds, asdescribed herein.

Any suitable carrier can be utilized. Suitable carriers includepharmaceutically or physiologically acceptable carriers. The followingmethods and carriers are merely exemplary and are in no way limiting. Inthe case of oral preparations, a compound of the present invention canbe administered alone or in combination with a therapeutically effectiveamount of at least one other compound. The active ingredient(s) can becombined, if desired, with appropriate additives to make tablets,powders, granules, capsules, or the like.

Suitable additives can include, for example, lactose, mannitol, cornstarch or potato starch. Suitable additives also can include binders,for example, crystalline cellulose, cellulose derivatives, acacia, cornstarch, or gelatins; disintegrants, for example, corn starch, potatostarch or sodium carboxymethylcellulose; with lubricants such as talc ormagnesium stearate. If desired, other additives such as, for example,diluents, buffering agents, moistening agents, preservatives, and/orflavoring agents, and the like, can be included in the composition.

The compounds used in accordance with the present invention can beformulated into a preparation for injection by dissolution, suspension,or emulsification in an aqueous or nonaqueous solvent, such as vegetableoil, synthetic aliphatic acid glycerides, esters of higher aliphaticacids, or propylene glycol (if desired, with conventional additives suchas solubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers, and preservatives). The compounds of the present inventionalso can be made into an aerosol formulation to be administered viainhalation. Such aerosol formulations can be placed into pressurizedacceptable propellants such as dichlorodifluoromethane,fluorohydrocarbons, propane, nitrogen, and the like.

The compounds of the present invention can be formulated intosuppositories by admixture with a variety of bases such as emulsifyingbases or water-soluble bases. The suppository formulations can beadministered rectally, and can include vehicles such as cocoa butter,carbowaxes, and polyethylene glycols, which melt at body temperature,but are solid at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions can be provided wherein each dosage unit, e.g.,teaspoonful, tablespoonful, tablet, or suppository contains apredetermined amount of the composition containing the compound of thepresent invention. Similarly, unit dosage forms for injection orintravenous administration can comprise a composition as a solution insterile water, normal saline, or other pharmaceutically acceptablycarrier.

The term “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of at least one compound orcompounds of the present invention (alone or, if desired, in combinationwith another therapeutic agent). The unit dosage can be determined bymethods known to those of skill in the art, for example, by calculatingthe amount of active ingredient sufficient to produce the desired effectin association with a pharmaceutically acceptable carrier. Thespecifications for the unit dosage forms that can be used in accordancewith the present invention depend on the particular effect to beachieved and the particular pharmacodynamics associated with thecompound(s) in the individual host.

Pharmaceutically acceptable carriers, for example, vehicles, adjuvants,excipients, or diluents, are accessible to those of skill in the art andare typically available commercially. One skilled in the art can easilydetermine the appropriate method of administration for the exactformulation of the composition being used. Any necessary adjustments indose can be readily made by a skilled practitioner to address the natureor severity of the condition being treated. Adjustments in dose also canbe made on the basis of other factors such as, for example, theindividual patient's overall physical health, sex, age, prior medicalhistory, and the like.

The dose administered to a mammal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the mammal over a reasonable time frame. The dose will bedetermined by the strength of the particular composition employed(taking into consideration, at least, the bioactivity of anydecomposition products derived from the compounds) and the condition ofthe mammal (e.g., human), as well as the body weight of the mammal(e.g., human) to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse sideeffects that might accompany the administration of a particularcomposition. A suitable dosage for internal administration is 0.01 to100 mg/kg of body weight per day, such as 0.01 to 35 mg/kg of bodyweight per day or 0.05 to 5 mg/kg of body weight per day. A suitableconcentration of the conjugate in pharmaceutical compositions fortopical administration is 0.05 to 15% (by weight), preferably 0.02 to5%, and more preferably 0.1 to 3%.

The compounds of the present invention can be utilized in a variety oftherapeutic and non-therapeutic applications. It will be appreciatedthat one or more compounds of the present invention can be used, forexample, as a control in diagnostic kits, bioassays, or the like.Preferably, the method of the present invention is appliedtherapeutically, for example, toward the treatment or prevention ofcancer or toward the treatment or prevention a condition (e.g., anabnormal condition or a disease) treatable by the inhibition ofvacuolar-type (H+)-ATPase. The compound(s) of the present invention canbe administered alone, or in combination with a therapeuticallyeffective amount of at least one additional compound other than acompound of the present invention.

Accordingly, the present invention further provides a method of treatingor preventing a condition treatable by the inhibition of vacuolar-type(H+)-ATPase, which method includes administering to a patient avacuolar-type (H+)-ATPase-inhibiting amount of at least one compound ofthe present invention. More particularly, the present invention providesa method of treating or preventing a condition treatable by theinhibition of vacuolar-type (H+)-ATPase, which method includesadministering a vacuolar-type (H+)-ATPase inhibiting-effective amount ofat least one compound of formula (I). By “prophylactic” is meant anydegree in inhibition of vacuolar-type (H+)-ATPase or inhibition of theonset of cancer, including complete inhibition. By “therapeutic” ismeant any degree in inhibition of vacuolar-type (H+)-ATPase orinhibition of the growth or metastasis of the cancer in the mammal(e.g., human).

A number of conditions can be treated in accordance with the method ofthe present invention. The vacuolar-type (H+)-ATPase inhibitingcompounds and compositions of the present invention can be usedmedically to regulate biological phenomena including, but not limitedto: intra-organellar acidification of intracellular organelles; urinaryacidification; bone resorption; fertility; angiogenesis; cellularinvasiveness (e.g., tumor cell invasiveness); tumor cell proliferationand metastasis; and the development of drug resistance in tumor cells.The compounds of the present invention are therefore useful in thetreatment of diseases that can be controlled by the inhibition ofvacuolar-type (H+)-ATPase. Such diseases include, for example,osteoporosis (see, e.g., Keeling et al., Ann. New York Acad. Sci., 834,600-608 (1997)), Alzheimer's disease, glaucoma, and abnormal urinaryacidification (see, e.g., Nelson, TIPS, 12, 71-75 (1991)). Moreover, thevacuolar-type (H+)-ATPase inhibitors of the present invention can beused in the treatment or prevention of diseases which utilize anacid-promoted cell penetration mechanism. For example, the compounds ofthe present invention can be used to inhibit the entry of viruses (e.g.,baculoviruses and retroviruses), or to inhibit the entry of proteintoxins (e.g., diphtheria toxin), into cells (see, e.g., Mellman et al.,Ann. Rev. Biochem., 55, 663-699 (1986)). The compounds of the presentinvention also can be used to inhibit fertility in an animal, forexample, a human (see, e.g., Wassarman, Science, 235, 553-560 (1987)),or to inhibit the proliferation, invasiveness or metastasis of tumorcells, or to promote the sensitivity of cancer toward drugs byinhibiting the ability of cancer cells to develop resistance to drugs,thereby facilitating and/or making possible the chemotherapeutictreatment of cancer (see, e.g., Marquardt and Center, J. Natl. CancerInst., 83, 1098-1102 (1991)).

Thus, as indicated above, the method of the present invention include amethod of treating conditions selected from the group consisting ofosteoporosis, Alzheimer's disease, glaucoma, fertility, abnormal urinaryacidification, abnormal secretion of degradative enzymes, and cancer. Inaccordance with method of the present invention, it is preferred that avacuolar-type (H+)-ATPase inhibiting-effective amount is used. In thatregard, it is preferred that the vacuolar-type (H+)-ATPaseinhibiting-effective amount is effective to inhibit one or moreconditions selected from the group consisting of intra-organellaracidification of intracellular organelles, urinary acidification, boneresorption, fertility, drug-resistance of tumor cells, tumor cellproliferation, cellular invasiveness, angiogenesis, and metastasis.

The method of the present invention further includes administering avacuolar-type (H+)-ATPase inhibiting-effective amount of at lease oneadditional compound other than a compound of the present invention,e.g., a compound other than a compound of formula (I). In someinstances, the method of the present invention can be made moreeffective by administering one or more other vacuolar-type (H+)-ATPaseinhibitors (e.g., a concanamycin and/or a bafilomycin and/orbenzolactone enamide, such as a salicylihalamide or a lobatamide), alongwith a compound of the present invention. One or more compounds of thepresent invention also can be co-administered in combination with ananticancer agent other than a compound of the present invention, forexample, to inhibit the development of cancer cell resistance to theanticancer agent.

In accordance with the method of the present invention, one or morecompounds of the present invention can be administered by any suitableroute including, for example, oral administration, intramuscularadministration, subcutaneous, intravenous administration, or the like.For example, one or more vacuolar-type (H+)-ATPase inhibitors of thepresent invention (or a composition thereof) can be administered as asolution that is suitable for intravenous injection or infusion, atablet, a capsule, or the like, or in any other suitable composition orformulation as described herein.

The vacuolar-type (H+)-ATPase “inhibiting-effective amount,” as utilizedin accordance with the composition and method of the present invention,includes the dose necessary to achieve a vacuolar-type (H+)-ATPase“inhibiting-effective level” of the active compound in an individualpatient. The vacuolar-type (H+)-ATPase inhibiting-effective amount canbe defined, for example, as that amount required to be administered toan individual patient to achieve a vacuolar-type (H+)-ATPaseinhibiting-effective blood level, tissue level, and/or intracellularlevel of a compound of the present invention to effect the desiredmedical treatment.

When the effective level is used as the preferred endpoint for dosing,the actual dose and schedule can vary depending, for example, uponinterindividual differences in pharmacokinetics, drug distribution,metabolism, and the like. The effective level also can vary when one ormore compounds of the present invention are used in combination withother therapeutic agents, for example, one or more additionalvacuolar-type (H+)-ATPase inhibitors, anticancer compounds, or acombination thereof. Moreover, the effective level can vary dependingupon the disease for which treatment is desired. For example, theeffective level for the treatment of osteoporosis may vary relative tothe effective level required for the treatment of abnormal urinaryacidification, or for the inhibition of fertility.

The unique vacuolar-type (H+)-ATPase inhibitory activity of thecompounds of the present invention can be determined using any suitablemethod known in the art, for example, assay methods. A suitable assaymethod for measuring vacuolar-type (H+)-ATPase inhibitory activity isdescribed, for example, in Chan et al., Anal. Biochem., 157, 375-380(1986). Alternatively, the unique vacuolar-type (H+)-ATPase inhibitoryactivity of the compounds of the present invention can be demonstratedusing the U.S. National Cancer Institute's (NCI)'s 60 cell-line, humantumor, disease-oriented screen, which can accurately predict theanticancer activity of chemical compounds. Significantly, the NCI 60cell-line screen also is a powerful tool that can be used to predictother types of biological activity, not limited to anticancer activity.In particular, the NCI 60 cell-line screen can be used to accuratelypredict antitumor activity as well as vacuolar-type (H+)-ATPaseinhibitory activity (see Boyd, PCT International Patent Application No.PCT/US00/05582).

Irrespective of vacuolar-type (H+)-ATPase inhibitory activity, thecompounds of the present invention have anticancer activity against anumber of different cancer cell lines, including human cancers, asdemonstrated in the NCI 60 cell-line screen. Exemplary compounds of thepresent invention possess potent antitumor activity (see, e.g., Example3). To the extent that the compounds used in accordance with the presentinvention have anticancer activity, the effective blood level can bedetermined by analogy based on the effective blood level correspondingto anticancer activity. As indicated above, the NCI 60 cell-line humantumor screen measures the ability of a compound to selectively kill orinhibit the growth of diverse human cancers. Using this screen, it isshown that the compounds of the present invention are highly activeagainst certain types of human solid tumors (e.g., non-small cell lungcancer, renal cancer, and melanoma), which are very resistant orcompletely resistant to existing anticancer drugs. It is also shown thatthe compounds of the present invention are active against many othertypes of human solid tumors and leukemia cancer cells. By theseobservations, and with other detailed analyses of tumor cellularresponse profiles, it can be demonstrated that the compounds of thepresent invention are novel anticancer agents having considerablepromise, for example, as therapeutic agents for the treatment of humansolid tumors.

The compounds of the present invention are thus new and broadlyefficacious anticancer agents, which inhibit or destroy human leukemias,lymphomas, melanomas and solid tumors. Solid tumors may include lungcancer (e.g., non-small cell lung cancer), colon cancer, CNS cancer(e.g., brain cancer), melanoma, ovarian cancer, renal cancer, prostatecancer, head and neck cancer, testicular cancer, germ-line cancers,endocrine tumors, uterine cancer, breast cancer, sarcomas, gastriccancer, hepatic cancer, esophageal cancer, pancreatic cancer, and thelike. Preferably the cancer is colon cancer, melanoma, breast cancer,ovarian cancer or non-small lung cancer.

The need for new classes of anticancer drugs remains an urgent worldwidepriority, which is being addressed effectively through new research anddevelopment applications of the NCI 60 cell-line screen. Reviews can befound, for example, in Boyd and Paull, Drug Dev. Res., 34, 91-109(1995); Weinstein et al., Science, 275, 343-349 (1997); and Greyer andChabner, In: Cancer: Principles and Practice of Oncology, 5th Ed.(DeVita, V. T., et al., eds.), Philadelphia: Lippincott-Raven, 1977, pp.385-394. The NCI screen provides an unprecedentedly rich informationcontent to support the identification of important new classes ofanticancer drugs. For example, see Weinstein et al., Science, 275,343-349 (1997); Greyer and Chabner, In: Cancer: Principles and Practiceof Oncology, 5th Ed. (DeVita, V. T., et al., eds.), Philadelphia:Lippincott-Raven, 1977, pp. 385-394; and Sausville, In: Anticancer DrugDevelopment Guide: Preclinical Screening, Clinical Trials, and Approval(Teicher, B. A., ed.), Totowa, N.J.: Humana Press, Inc., 1997, pp.217-226.

Accordingly, the present invention further provides a method ofpreventing or treating cancer, which method comprises administering ananticancer effective amount of at least one compound of the presentinvention. The anticancer effective amount can be determined by methodsknown in the art including, for example, by determining an amount to beadministered effective to produce an “effective level” in the subjectpatient. The effective level can be chosen, for example, as that level(e.g., 10⁻¹¹−10⁻⁷ M) effective to inhibit the proliferation of tumorcells in a screening assay. Similarly, the effective level can bedetermined, for example, on the basis of the blood or tissue level in apatient that corresponds to a concentration of a therapeutic agent thateffectively inhibits the growth of human cancers in an assay that isclinically predictive of anticancer activity. Further, the effectivelevel can be determined, for example, based on a concentration at whichcertain markers of cancer in a patient's blood are inhibited by aparticular compound that inhibits cancer. Alternatively, the effectivelevel can be determined, for example, based on a concentration effectiveto slow or stop the growth of a patient's cancer, cause a patient'scancer to regress or disappear, render a patient asymptomatic to aparticular cancer, or improve a cancer patient's subjective sense ofcondition. The anticancer effective level can then be used toapproximate (e.g., by extrapolation), or even to determine, the level,which is required clinically to achieve a vacuolar-type (H+)-ATPaseinhibiting-effective blood, tissue, and/or intracellular level to effectthe desired medical treatment. It will be appreciated that thedetermination of the therapeutically effective amount clinicallyrequired to effectively inhibit vacuolar-type (H+)-ATPase activityrequires consideration of other variables that can influence theeffective level, as discussed herein. When a fixed effective amount isused as a preferred endpoint for dosing, the actual dose and dosingschedule for drug administration can vary for each patient dependingupon factors that include, for example, inter-individual differences inpharmacokinetics, drug disposition, metabolism, whether other drugs areused in combination, or other factors described herein that effect theeffective level.

One skilled in the art can readily determine the appropriate dose,schedule, or method of administering a particular formulation, in orderto achieve the desired effective level in an individual patient. Oneskilled in the art also can readily determine and use an appropriateindicator of the effective level of the compounds of the presentinvention. For example, the effective level can be determined by directanalysis (e.g., analytical chemistry) or by indirect analysis (e.g.,with clinical chemistry indicators) of appropriate patient samples(e.g., blood and/or tissues). The effective level also can bedetermined, for example, by direct or indirect observations such asurine acidity, change in bone density, decrease in ocular pressure, orby the shrinkage or inhibition of growth of a tumor in a cancer patient(e.g., if the compound in question has anticancer activity). There aremany references in the art that describe the protocols used inadministering active compounds to a patient in need thereof. Forexample, the protocols used in the administration of anticancer agentsto patients are described in “Cancer Chemotherapy: Principles andPractice” ed., Chabner and Collins, J. B. Lippincott, 1990, especiallychapter 2, by J. B. Collins. See also Boyd, WO 99/05136.

The present inventive method of preventing or treating cancer furtherincludes administering an anticancer effective amount of at least oneadditional compound other than a compound of the present invention, forexample, a compound other than a compound of formula (I). For example,one or more compounds of the present invention can be co-administeredwith an anticancer agent, in which case the effective level desirably isthe level needed to inhibit the ability of the cancer to developresistance to the anticancer agent. Suitable anticancer compoundsinclude, for example, all of the known anticancer compounds approved formarketing in the United States, and those that will become approved inthe future, for which drug resistance thereto can be controlled by theinhibition of vacuolar-type (H+)-ATPase.

The demonstration of antitumor, vacuolar-type (H+)-ATPase-inhibitory andother biological activities is based on the correlation of activitypatterns generated in the NCI screen by compounds having known activity.The compounds compared in the correlation need not have particularlypotent anticancer activity in order to display an activity patternsuitable for correlation in the NCI screen. Interestingly, compoundsneed not be structurally similar to one another in order correlate witheach other in the NCI screen. Even if two structurally dissimilarcompounds correlate strongly with each other in the NCI screen, they canbe accurately predicted to have the same biological activity as eachother in virtually any application, including non-cancer applications.For reviews pertinent to the NCI 60 cell-line screen, see Boyd, In:Current Therapy in Oncology (Niederhuber, ed.), Philadelphia: B. C.Decker, Inc., 1993, pp. 11-22; Boyd and Paull, Drug Dev. Res., 34,91-109 (1995); Greyer and Chabner, In: Cancer Principles and Practice ofOncology, 5th Ed. (DeVita et al., eds.), Philadelphia: Lippincott-Raven,1977, pp. 385-394; Paull et al., In: Cancer Chemotherapeutic Agents(Foye, ed.), Washington, D.C.: American Chemical Society Books, 1995,pp. 9-45; and Weinstein et al., Science, 275, 343-349 (1997).

The NCI 60 cell-line human tumor screen measures the ability of acompound to selectively kill or inhibit the growth of diverse humancancers. Generally, in the NCI screen, the compounds of the presentinvention display potent activity against certain types of human solidtumors (e.g., non-small cell lung cancer, renal cancer, and melanoma),and resistant strains thereof. By these observations, and with otherdetailed analyses of the characteristic tumor cellular responseprofiles, it can be shown that the compounds of the present inventionhave a uniquely characteristic bioactivity profile.

The NCI 60 cell-line human tumor primary screen also provides a means bywhich to identify natural sources of compounds. The NCI screen wasdesigned and implemented during 1985-1990 under the direction, closescrutiny, and supervision of several internationally comprised andrenowned extramural (non-NCI) advisory and review groups, including theNCI Division of Cancer Treatment's Board of Scientific Counselors, an AdHoc Expert Review-Committee thereof, the National Cancer Advisory Board,and the President's Cancer Panel (see Boyd, In: Anticancer DrugDevelopment Guide: Preclinical Screening, Clinical Trials, and Approval(Teicher, B. A., ed.), Totowa, N.J.: Humana Press, Inc., pp. 23-42,1997). The impetus for development of the NCI screen was theinternational recognition that most of the commercially availableanticancer drugs worldwide are essentially inactive or only transientlyactive against most forms of human cancer. Reviews are disclosed, forexample, in Boyd, In: Cancer: Principles and Practice of OncologyUpdates (DeVita, V. T., Jr., et al., eds), Philadelphia: Lippincott,1989, pp. 11-22; and Boyd, In: Current Therapy in Oncology (Niederhuber,J. E., ed.), Philadelphia: BC Decker, 1993, pp. 11-22. Although this NCIscreen has been operational only since 1990, it has already led to thediscovery, development, and clinical use of significant new anticancerdrugs in human cancer patients. For example, see Weinstein et al.,Science, 275, 343-349 (1997); Greyer and Chabner, In: Cancer: Principlesand Practice of Oncology, 5th Ed. (DeVita, V. T., et al., eds.),Philadelphia: Lippincott-Raven, 1977, pp. 385-394; and Sausville, In:Anticancer Drug Development Guide: Preclinical Screening, ClinicalTrials, and Approval (Teicher, B. A., ed.), Totowa, N.J.: Humana Press,Inc., 1997, pp. 217-226.

The NCI screen consists of a panel of 60 different human tumor celllines against which compounds are tested over a defined range ofconcentrations to determine the relative degree of growth inhibition orcytotoxicity against each cell line. The design and operation of thescreen is such that for each compound tested, both the absolute andrelative sensitivities of individual cell lines comprising the screenare sufficiently reproducible that a characteristic profile or“fingerprint” of cellular response is generated. Compounds that areactive in the NCI screen show pronounced differential tumorgrowth-inhibitory and/or cytotoxic effects to the diverse cell linescomprising the 60 cell-line panel. The degree of differential responsebetween the most and least sensitive lines typically may be relativelysmall (e.g., 2- to 10-fold), or occasionally as great as 3-4 orders ofmagnitude. Furthermore, the cell lines may be widely heterogeneous inresponse to a given compound, or they may be comparatively homogeneous,with only a relatively few lines showing much greater or lessersensitivity than average. Regardless of the magnitude of thedifferential or the degree of heterogeneity of response of the cell linepanel, it is the reproducibility of the response “fingerprint” that isimportant to the useful information contained therein.

Detailed disclosures of the screening assay are published, for example,in Monks et al., J. Natl. Cancer Inst., 83, 757-766 (1991); Skehan etal., J. Natl. Cancer Inst., 82, 1107-1112 (1990); and Boyd and Paull,Drug Dev. Res., 34, 484-488 (1995). The identities, sources, derivation,morphological, and immunocytochemical characteristics, and methods ofmaintenance of the cell lines comprising the NCI 60 cell line panel havebeen described in detail, for example, in Boyd, In: Cancer: Principlesand Practice of Oncology Updates (DeVita, V. T., Jr., et al., eds),Philadelphia: Lippincott, 1989, pp. 1-12; Monks et al., J. Natl. CancerInst., 83, 757-766 (1991); Stinson et al., Anticancer Res., 12,1034-1035 (1992); and Boyd and Paull, Drug. Dev. Res., 34, 91-109(1995).

In the screening assay, each agent is tested over a broad concentrationrange against every cell line in the panel. All lines are inoculatedonto a series of standard 96-well microtitre plates on day zero,followed by a 24 h incubation in the absence of the test compound. Theinoculation densities employed depend upon the particular cell line andits growth characteristics. Inoculation densities used are as publishedin Monks et al., J. Natl. Cancer Inst., 83, 757-766 (1991); and Boyd andPaull, Drug Dev. Res., 34, 91-109 (1995). Test compounds are evaluatedat five 10-fold dilutions. Following a 48-hour incubation with the testcompound, the cells are assayed by the sulforhodamine B procedure asdescribed in Skehan et al., J. Natl. Cancer Inst., 82, 1107-1112 (1990);Monks et al., J. Natl. Cancer Inst., 83, 757-766 (1991); and Rubinsteinet al., J. Natl. Cancer Inst., 82, 1113-1118 (1990). Optical densitiesare measured on automated plate readers, followed by computerized dataacquisition, processing, storage, and availability for display andanalysis. Each successful test of a compound generates 60 dose-responsecurves, which are printed in the NCI screening data report as a seriesof composites comprising the tumor-type subpanels. Data for anyindividual cell line(s) failing quality control criteria, or otherwisedeficient for any cell line(s) not tested successfully, are eliminatedfrom further analysis and are deleted from the screening report.

The “percentage growth” (PG) term, and meaning of the +50, 0, and −50response reference lines, the calculated response parameters, GI₅₀, TGI,and LC₅₀, construction and use of “mean-graphs” and the COMPAREpattern-recognition algorithms are briefly summarized as follows. The50% growth inhibition parameter (GI₅₀) is the concentration of test drugwhere 100×(T−T_(o))/(C−T_(o))=50=PG. The optical density of the testwell after the 48 hour drug exposure is T; the optical density at timezero is T_(o); and the control optical density is C. The PG is aT/C-like parameter that can have values from +100 to −100. Whereas theGI₅₀ may be viewed as a growth-inhibitory level of effect, the TGIsignifies a “total growth inhibition” or cytostatic level of effect. TheTGI is the drug concentration where 100×(T−T_(o))/(C−T)=0=PG. The LC₅₀is the lethal concentration, “net cell killing” or cytotoxicityparameter. It is the concentration where 100×(T−T_(o))/T_(o)=−50=PG. Thecontrol optical density is not used in the calculation of LC₅₀. For adetailed description of the “percentage growth” (PG) term, the +50, 0,and −50 response reference lines, the calculated response parameters,GI₅₀, TGI, and LC₅₀, the construction and use of “mean-graphs,” and theCOMPARE pattern-recognition algorithms, see Boyd et al., In: CytotoxicAnticancer Drugs: Models and Concepts for Drug Discovery and Development(Valeriote, F. A., et al., eds.), Amsterdam: Kluwer Academic Publishers,1992, pp. 11-34; Monks et al., J. Natl. Cancer Inst., 83, 757-766(1991); and Boyd and Paull, Drug Dev. Res., 34, 91-109 (1995).

A mean-graph is a pattern created by plotting positive and negativevalues, termed “deltas,” generated from a set of GI₅₀, TGI, or LC₅₀concentrations obtained for a given compound tested against each cellline in the NCI in vitro screen. The deltas are generated from the GI₅₀,TGI, or LC₅₀ data by a three-step calculation. For example, the GI₅₀value for each cell line successfully tested against a given compound isconverted to its log₁₀ GI₅₀ value. The mean panel log₁₀ GI₅₀ value isobtained by averaging the individual log₁₀ GI₅₀ values. Each log₁₀ GI₅₀value then is subtracted from the panel mean to create the correspondingdelta.

To construct the mean-graph, the deltas are plotted horizontally inreference to a vertical line that represents the calculated mean panelGI₅₀. The negative deltas are plotted to the right of the mean referenceline, thereby proportionately representing cell lines more sensitivethan the calculated average. Conversely, the positive deltas are plottedto the left of the reference line to represent the less sensitive celllines to the given agent. Thus, for example, a bar projecting 3 units tothe right of the vertical reference line in a GI₅₀ mean-graph indicatesthat the GI₅₀ concentration for that cell line is 1000 times less thanthe panel-averaged GI₅₀ concentration. The TGI and LC₅₀ mean-graphs areprepared and interpreted similarly.

Three additional numbers are printed at the base of each of the threerespective mean-graphs. These numbers are the MG-MID, the Delta (not beconfused with the “delta” for an individual cell line), and the Range.The MG-MID is the calculated mean panel GI₅₀, TGI, or LC₅₀. The Delta isthe number of log₁₀ units by which the delta of the most sensitiveline(s) of the panel differ(s) from the corresponding MG-MID. Similarly,the Range is the number of log₁₀ units by which the delta of the mostsensitive line(s) of the panel differ(s) from the delta(s) of the leastsensitive line(s).

COMPARE is a computerized, pattern-recognition algorithm used in theevaluation and exploitation of data generated by the NCI screen. Inessence, COMPARE is a method of determining and expressing the degree ofsimilarity, or lack thereof, of mean-graph profiles generated on thesame or different compounds. An early impetus for the creation of such atool during the development of the screen was the need to standardizeand to establish and monitor the screen's consistency andreproducibility over time. This is accomplished by the regular testingof standard compounds that are expected to generate the same or verysimilar profiles when screened repetitively against the same panel ofcell lines.

The NCI screen is repetitively calibrated. In the course ofstandardizing the screen, NCI selected as reference compoundsapproximately 170 agents for which a considerable amount of informationwas available about their preclinical and/or clinical anticancerproperties and mechanism(s) of action. These compounds includedcommercially marketed anticancer drugs, investigational anticancerdrugs, and other anticancer drugs which were or had been in preclinicaldevelopment based upon activities in other cancer-related test systems.The repetitive periodic screening of these prototype “standard agents”(the cumulative compilation of results of which forms the “StandardAgents Database”) remains the basis for calibration and standardizationof the screen.

Significantly, the NCI's Standard Agent Database also provides a key tomany useful new drug discovery applications. For example, thecharacteristic response profile “fingerprint” of a selected standardagent may be used as the “seed” to probe any other available mean-graphdatabase to see if there are any closely matching profiles containedtherein. Similarly, a profile selected from any available mean-graphdatabase can be used to probe the “Standard Agent Database” to determinewhether or not there are any closely matching standard agent profiles.Additional databases used for such studies may be constructed or definedas desired and may be relatively small (e.g., comprising a singlecompound or a selected congeneric series of compounds) or very large(e.g., the entire databases from all pure compounds, mixtures,fractions, and extracts tested in the NCI screen to date).

Initial NCI studies with COMPARE showed that compounds with matchingmean-graph patterns often had related chemical structures. However,closer examination of this phenomenon revealed that certain compounds ofunrelated structures had matching mean-graph patterns and shared thesame or related biochemical mechanisms of action. For example, see Boyd,In: Current Therapy in Oncology (Niederhuber, J. E., ed.), Philadelphia:B C Decker, 1993, pp. 11-22; and Paull et al., In: Cancer TherapeuticAgents, Washington, D.C.: Am. Chem. Soc. Books, pp. 9-45 (1995); andreferences cited therein. COMPARE analyses can be performed using themean-graph deltas calculated from either the GI₅₀'s, the TGI's, or theLC₅₀'s. When a selected particular mean-graph profile or “seed” is usedto probe a given database, the appropriate delta value for each cellline is compared to the corresponding delta value for the same cell linefor every mean-graph entry in the specified database set. If eitherdelta value is missing for any cell line (e.g., due to test failure orquality control deletion), then that cell line is eliminated entirelyfrom the calculation for that particular seed/mean-graph anddatabase/mean-graph pair. Thus, for each mean-graph in the specifieddatabase, a set of pairs (maximum of 60) of delta values is obtained.The commercially available SAS statistical program is used to calculatea Pearson product moment correlation coefficient (0.0-1.0) for each setof delta value pairs. The mean-graphs of all compounds in the specifieddatabase can then be rank-ordered for similarity to the seed mean-graph.Public access to the NCI's “Standard Agents Database,” as well as to avariety of NCI screening data display and analysis tools, includingCOMPARE, are available to investigators worldwide via the Internet(http://dtp.nci.nih.gov/).

By regular application of COMPARE, using selected prototype seedcompounds from the Standard Agents Database, NCI has maintained ongoingsurveillance of the total historical screening database accrued frominception to date. In this manner, compounds with screening fingerprintsmatching standard agent(s) having known or presumed known mechanism(s)of actions can be identified. NCI has been able to associate andsubsequently confirm the database classification of compounds ofpreviously unknown mechanisms of action into a number of different knownmechanistic classes of interest. For example, new members have beenclassified within general mechanistic categories of tubulin-interactiveantimitotics, antimetabolites, alkylating agents, topoisomeraseinhibitors, DNA binders, and the like. These and numerous other examplesresulting from this kind of database prospecting have been published,for example, in Paull et al., Cancer Res., 52, 3892-3900 (1992), andreferences cited therein; and Paull et al., In: Cancer ChemotherapeuticAgents, Washington, D.C.: Am. Chem. Soc. Books, 1995, pp. 9-45, andreferences cited therein.

Quite surprisingly, it has been discovered that, uniquely among the tensof thousands of mean-graph “fingerprints” analyzed by applicants, thecharacteristic screening “fingerprints” for certain exemplary compoundsof the present invention correlate almost perfectly with those ofprotypical vacuolar-type (H+)-ATPase inhibitory compounds, concanamycinA, bafilomycin A1, salicylihalamide A and lobatamide A, all of which arestructurally unrelated to the compounds of the present invention. Thecorrelation for certain exemplary compounds of the present invention isso precise, that the possibility of coincidence is effectively ruledout. It is therefore concluded that the compounds of the presentinvention, whose mean graph fingerprints in the NCI screen correlatehighly with those of concanamycin A, bafilomycin A1, salicylihalamide A,and lobatamide A, are inhibitors of vacuolar-type (H+)-ATPase. Indeed,it can readily be demonstrated by specific vacuolar-type (H+)-ATPasebioassay that compounds of the present invention whose fingerprints inthe NCI 60 cell-line screen correlate with those of the structurallyunrelated but known vacuolar-type (H+)-ATPase inhibitors (e.g., seeBoyd, PCT International Patent Application No. PCT/US00/05582)concanamycin A, bafilomycin A1, salicylihalamide A and lobatamide A havepotent vacuolar-type (H+)-ATPase inhibitory activity, as expected. Thus,the NCI 60 cell-line screen can be used to demonstrate that any selectedcompound of the present invention is an inhibitor of vacuolar-type(H+)-ATPase.

Compounds whose mean-graph “fingerprints” generated by the NCI 60cell-line screen correlate highly with one another can be expected toshare a common molecular target or biological mechanism of action, evenif the compounds differ significantly in structure. A high correlationcan be established, for example, by COMPARE correlation coefficients ofapproximately 0.8 to 0.9, or greater. See Boyd, In: Current Therapy inOncology (Niederhuber, J. E., ed.) Philadelphia: B. C. Decker, 1993, pp.11-22; Boyd and Paull, Drug Dev. Res., 34, 91-109, 1995; Paull et al.,In: Cancer Therapeutic Agents, Washington, D.C.: Am. Chem. Soc. Books,1995, pp. 9-45. Thus, the concanamycins, bafilomycins, salicylihalamidesand lobatamides, and exemplary compounds of the present invention, forexample, whose NCI 60 cell-line screen correlation coefficients withrespect to each other are high, can all be shown to share the samemolecular target, vacuolar-type (H+)-ATPase. Further illustration ofthis characteristic is provided in Example 4.

One skilled in the art will appreciate that not all vacuolar-type(H+)-ATPase inhibitors will inhibit equally the vacuolar-type(H+)-ATPase activity present in different kinds or locations ofintracellular organelles, or in different kinds or locations of plasmamembranes, or in different kinds or locations of cells or tissues. Inother words, a given vacuolar-type (H+)-inhibitory compound maypreferentially inhibit vacuolar-type (H+)-ATPase activity in one or morekind or location of intracellular organelle, plasma membrane, cell ortissue. Thus, the skilled practitioner will typically select aparticular vacuolar-type (H+)-ATPase inhibitory compound for a desiredtherapeutic use. Compound selection can be based upon the particularkind or location of intracellular organelle or plasma membranevacuolar-type (H+)-ATPase preferentially inhibited by the compound.Indeed, there are clear precedents in the literature to indicate thatcompounds can be selected for particular applications based uponpreferential inhibition of one or more kind of vacuolar-type (H+)-ATPaseover another. For example, Gagliardi et al., J. Med. Chem., 41,1568-1573, (1998), identified compounds that selectively inhibit humanosteoclast vacuolar-type (H+)-ATPase activity compared to human renalcortical vacuolar-type (H+)-ATPase activity; such compounds thereforeare expected to be particularly useful in treating osteoporosis.

In addition to the pharmacological utility of inhibitors of mammalianvacuolar-type (H+)-ATPase activity, pharmacological utility may also beobtained by inhibition of non-mammalian vacuolar-type (H+)-ATPaseactivity. For example, the known vacuolar-type (H+)-ATPase inhibitorsbafilomycin A₁ and concanamycin A potently inhibit fungal as well asmammalian vacuolar-type (H+)-ATPase activity, and those compounds havestrong antifungal activity. See Bowman et al., Proc. Natl. Acad. Sci.USA, 85, 7972-7976 (1988); Dröse et al., Biochemistry, 32, 3902-3906(1993); Dröse and Altendorf, J. Exp. Biol., 200, 1-8 (1997).

There is also evidence that vacuolar-type (H+)-ATPase plays importantroles in the proliferation of tumor cells, and the consequentinvasiveness and metastasis thereof. See Montcourrier et al., J. CellSci., 107, 2381-2391 (1994); Martinez-Zaguilan et al., Am. J. Physiol.,265, C1015-C1-29 (1993); Martinez-Zaguilan et al., J. Cell Physiol.,176, 196-205 (1998); Nishihara et al., Biochem. Biophys. Res. Commun.,212, 255-262 (1995); Manabe et al., J. Cell Physiol., 157, 445-452(1993). Furthermore, acidification of intracellular organelles cancontribute to the sequestration and cellular efflux of conventionalanticancer drugs. See Marquardt and Center, J. Natl. Cancer Inst., 83,1098-1102 (1991); Benderra et al., Intl. J. Oncol., 12, 711-715 (1998);Mariyama et al., J. Biochem., 115, 213-218 (1994). Therefore,vacuolar-type (H+)-ATPase inhibitory compounds of the present inventioncan be used to inhibit the proliferation of tumor cells, as well as theconsequent invasiveness and metastasis thereof. Furthermore, thecompounds of the present invention can be used to inhibitdrug-resistance of tumor cells to conventional anticancer agents.

The particular compound or composition used in accordance with thepresent invention may be selected based upon the desired kind or site ofvacuolar-type (H+)-ATPase inhibition, and/or based upon otherpharmacological, toxicological, pharmaceutical or other pertinentconsiderations that are well-known to those skilled in the art. Routinemethods for the specific bioassay, quantitation and comparisons ofinhibitory activity of compounds and compositions of the presentinvention against vacuolar-type (H+)-ATPase activity in various tissues,cells, organelles and other preparations is well-documented in theliterature (see, e.g., Bowman et al., Proc. Natl. Acad. Sci. USA, 85,7972-7976 (1988); Gagliardi et al., J. Med. Chem., 41, 1883-1893 (1998);Gagliardi et al., J. Med. Chem., 41, 1568-1573 (1998); and referencescited therein).

COMPARE analyses of GI₅₀ and TGI mean-graph screening profiles ofcertain compounds of the present invention can be consistently shown tohave a high degree of commonality with respect to each other (e.g., GI₅₀and TGI-COMPARE Pearson correlation coefficients of at least 0.6-0.8 orgreater), but do not show any such correlations with any known standardagent. Similarly, extracts of natural organisms, which can be shown tocontain compounds of the present invention, typically give GI₅₀ and TGImean-graph screening fingerprints with similarly high GI₅₀ andTGI-COMPARE Pearson correlations (e.g., typically 0.6-0.7 or greater) tothe compounds of the present invention. This allows a person of skill inthe art to readily identify productive source organisms and extractsthereof, from which the skilled artisan can readily obtain and use thecompounds of the present invention or precursors thereof. Identificationand/or characterization of the present inventive compounds is furtherfacilitated by the presence of certain characteristic NMR signals suchas described in Example 2. Such characteristic NMR signals can furtherconfirm the identification and selection of compound mixtures, includingcrude extracts of natural organisms and partially purified fractionsthereof, or synthetic or semi-synthetic reaction products, that containthe compounds.

Certain compounds of the present invention can be readily obtained fromnatural sources, including solvent extracts of marine sponges, forexample, from aqueous extracts of sponge species from the genusPoecillastra species. Extracts of Poecillastra species sponges can beprepared from any suitable solvent, for example, organic solvents,water, and mixtures thereof. Fresh sponges can be used, but moregenerally they are frozen immediately after harvesting, and then areeither used directly or are freeze-dried before the extraction is done.When a marine sponge is used as a source for obtaining compounds of thepresent invention, it is preferably from the genus Poecillastra species,but is more preferably a Poecillastra species, and is most preferably aPoecillastra species collected near Settlement Point, Grand BahamaIsland, Bahamas (see Example 1).

Specific extracts of Poecillastra species that contain compounds of thepresent invention can be identified and selected based upon theanticancer screening profile they produce in the NCI 60-cell human tumorscreen. Such extracts containing compounds of the present invention alsocan be identified and selected based upon key proton and carbon NMRsignals (e.g., see Table 1) that are characteristic of the structuralcomponent motif (I) shared by the compounds of the present invention(see also Example 1).

From the aforementioned selected extracts, a variety of methods can beused for isolation and purification of compounds of the presentinvention. During each step of isolation and purification, theaforementioned characteristic anticancer screening profile or a suitablebioassay, and the aforementioned characteristic proton NMR signals, canbe obtained for intermediate fractions, as well as partially purifiedand purified compounds, to ensure isolation of the desired compounds ofthe present invention.

A preferred method of obtaining certain compounds of the presentinvention or a precursor thereof from natural source materials includesthe steps of:

(a) obtaining a fresh or frozen sample of a marine sponge (or othersuitable natural source material) that includes one or more compounds ofthe present invention or a precursor thereof,

(b) extracting the sample with water and/or one or more organicsolvents, or mixtures thereof, which dissolves the compound(s) orprecursor(s) to form an extract,

(c) optionally treating the extract with a solvent (e.g., a nonsolventsuch as ethanol) to precipitate and remove high molecular weightproteins and sulfated polysaccharides,

(d) optionally partitioning the extract between an organic solvent andan aqueous solvent to form a partitioned organic solvent extract oraqueous solvent extract containing the desired compound(s) orprecursor(s),

(e) chromatographing, one or more times as necessary, the partitionedextract, for example, on an adsorption, partition, or reversed-phase, orsize-exclusion matrix, to produce one or more fractions, and

(f) isolating one or more compounds of the present invention or one ormore precursors thereof from one or more of the fractions.

In step (b), the solvent can include mixtures of suitable nonpolarorganic solvents or suitable polar organic solvents. Suitable nonpolarorganic solvents include, for example, CH₂Cl₂, CHCl₃, toluene, hexaneand the like. Suitable polar organic solvents include, for example,water, MeOH, EtOH, isopropyl alcohol, acetone and the like. In step (d)suitable organic nonpolar solvents include CH₂Cl₂, hexane, CCl₄, CHCl₃,MeOtBu, ethyl acetate and the like; and typical aqueous solvents caninclude, for example, mixtures of water and methanol. Non-limitingexamples of solvent mixtures that can be used optionally in thispartitioning step include: (1) CH₂Cl₂ and 19:1 H₂O-MeOH, (2) hexane and9:1 MeOH—H₂O, (3) CCl₄ and 8:2 MeOH—H₂O, (4) CH₂Cl₂ and 7:3 MeOH—H₂O,and (5) EtOAc and H₂O.

In step (e), the chromatography preferably is column chromatography.When column chromatography is used, the chromatographic matrixpreferably is the adsorption type, the partition type, thereversed-phase type, the size exclusion type, or a suitable combinationthereof. Preferably, the solvent and/or the matrix is not acidic innature when the compound to be isolated is not particularly acid stable.Sephadex™ LH-20, a particularly preferred matrix for isolation ofcertain types of compounds of the present invention, combines three ofthe aforesaid matrix types, and is characterized by mild treatment andgood recoveries.

The isolation step (f) can be carried out, for example, by evaporatingthe solvent, by recrystallization optionally after additionalconcentration using reversed-phase HPLC, or by using other isolationprocedures known in the art.

In a preferred isolation method, a selected sample of frozenPoecillastra species sponge is ground to a powder with dry ice. The dryice is allowed to sublime, distilled H₂O is added, and the thawedmaterial is stirred for 3 h at 3° C., and then centrifuged. The aqueoussupernatant is lyophilized and the concentrated extract is fractionatedon wide-pore reversed-phase C₄ media. The fraction eluting with MeOH—H₂O(2:1) is further separated on an LH-20 column using a MeOH:H₂O (7:3)solvent system. The early eluting material from this column isultimately purified by reversed-phase C₁₈ HPLC with a linear CH₃CN—H₂Ogradient (with or without the addition of about 0.01% trifluoroaceticacid) to give, after solvent removal, substantially purified compound(s)of the present invention.

The definitive proofs of structure of the isolated compounds can beobtained by a combination of methods including primary spectral analyses(e.g., high-resolution NMR and mass spectrometry, infrared and UVspectroscopy), comparisons of spectral and physico-chemical propertieswith related literature precedents, and by x-ray crystallographicanalysis. Various structural proofs are illustrated in Example 2 herein.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

High performance liquid chromatography (HPLC) was performed on aVarian-Rainin system employing a Dynamax C₁₈ column (1×25 cm), using aflow rate of 3 mL/minute and ultraviolet (UV) detection at 220 nm.Optical rotations were measured with a Perkin-Elmer 241 polarimeter. UVand infrared (IR) spectra were obtained on a Beckman DU-640 and aPerkin-Elmer 1600 FT-IR spectrometer, respectively. High resolution massspectra were acquired on a JEOL SX102 mass spectrometer.

Nuclear magnetic resonance (NMR) spectra were recorded at 29° C. on 800μg of poecillastrin A (1) dissolved in 180 μL of DMF-d₇ and placed in a3 mm NMR tube. The heteronuclear multi-bond connectivity (HMBC) datawere collected on a Varian INOVA 500 spectrometer outfitted with a 5 mmHCN inverse-detection Chili-Probe. The radio frequency coils andpreamplifier of the Chili-Probe were cooled with liquid helium toapproximately 25 K and 60 K, respectively. The HMBC, which was optimizedfor ¹J_(CH)=140 Hz and ^(n)J_(CH)=6 Hz, was acquired in approximately32.5 hrs with 1,025 (F2)×256 (F1) points (all data points reportedrepresent complex data points), 256 scans per increment, and F2 and F1spectral widths of 6,000 and 27,036, respectively. The F1 dimension waszero-filled to 2K and the data was processed with a sinebell weightingfunction. All other NMR data were obtained on a Varian INOVA 800spectrometer equipped with a 3 mm inverse-detection probe operated atroom temperature. An absolute value correlated spectroscopy spectrum(COSY) was acquired with 2K (F2)×192 (F1) points, 16 scans perincrement, and 8468 Hz spectral width in both dimensions. The totalcorrelation spectroscopy (TOCSY) data were acquired with a 60 ms mixingtime at 8.3 KHz field strength, 1K×224 points, 16 scans per increment,and 6,000 Hz spectral width in both dimensions. The rotating-frameOverhauser enhancement spectroscopy (ROESY) was acquired with a 300 msmixing time at 5.5 KHz field strength, 2K×256 points, 16 scans perincrement, and 8,468 Hz spectral width in both dimensions. Themultiplicity-edited HSQC, with ¹JCH=140 Hz, was acquired with 1,536×256points, 192 scans per increment, and spectral widths of 8,468 Hz in F2and 28,139 Hz in F1. The COSY data were processed with a sinebellweighting function and gaussian weighting was used for the other datasets obtained at 800 MHz. All of these experiments had the F2 dimensionzero-filled to 4K, except for the heteronuclear single quantumcorrelation (HSQC), which was zero-filled to 2K. The F1 dimension of theHSQC and COSY were zero-filled to 1K, while in the TOCSY and ROESY, F1was zero-filled to 512. Two- and three-fold linear predictions wereperformed on the HSQC and COSY data, respectively. Chemical shifts arereported in ppm relative to the residual nondeuterated solvent.Assignments for the ¹³C resonances were based on HSQC and HMBCcorrelations, and their multiplicities were inferred from themultiplicity-edited HSQC experiment.

Samples of the sponge Poecillastra species were collected by a mannedsubmersible at a depth of −359 meters near Settlement Point, GrandBahama Island, Bahamas. A voucher specimen (# Q66B974) for thiscollection is maintained at the Smithsonian Institution, Washington,D.C.

Example 1

This example describes the isolation and purification of poecillastrin A(1). The frozen Poecillastra sponge (394 g) was ground to a fine powderand extracted with H₂O to give 94 g of aqueous extract. After numerouschemical separation schemes were attempted, the followingbioassay-guided isolation procedure was employed. A 20 g portion of theextract was dissolved in 120 mL of H₂O and precipitated by addition of120 mL of ethanol (EtOH) and storage at −20° C. for 16 h. After removalof the precipitate by centrifugation, the supernatant was evaporated todryness and then partitioned between H₂O and normal-butanol (n-BuOH).The n-BuOH soluble material (1.03 g) was triturated with MeOH to give a917 mg methanol (MeOH)-soluble fraction. Separation of this material ona Sephadex LH-20 column eluted with MeOH—H₂O (7:3) provided a cytotoxicfraction (43 mg) that was chromatographed on a second Sephadex LH-20column eluted with 100% MeOH. The cytotoxic fractions from this LH-20column were combined to give 12.1 mg of material that was subjected toC₁₈ column chromatography eluted with MeOH—H₂O (2:3). Final purificationwas achieved by C₁₈ HPLC eluted with a linear gradient of CH₃CN—H₂O(9:11) to 100% CH₃CN (0.1% TFA vol/vol in the eluant) over 45 min togive 800 μg of poecillastrin A (1).

Example 2

This example demonstrates the structural proofs of particular compoundsof the present invention.

Poecillastrin A (1), such as obtained in Example 1, was a white gum withthe following spectral characteristics: UV [MeOH] λ_(max) 229 (ε10,800), 235 (ε 10,900), 263 (ε 10,200) nm; IR υ_(max) (neat on a NaClplate) 3500-3300, 1716, 1697, 1683, 1206, 1135 cm⁻¹; ¹H and ¹³C NMRdata, see Table 1; Fast atom bombardment mass spectrometry (FABMS)(M+Na)⁺ m/z 1464.9; High resolution fast atom bombardment massspectrometry (HRFABMS) of a CsI-doped sample, (M−H+2Cs)⁺ m/z 1706.7375,calcd for C₇₉H₁₃₀N₃O₂₀Cs₂, 1706.7350. An accurate determination of theoptical rotation was difficult due to the limited sample size. Forty-twoindividual measurements were made and all of them provided negativeoptical rotations. The optical rotation calculated for 1 was based onthe average of these forty two measurements: [α]²⁷ _(D) −8.3° (c 0.056,MeOH).

The molecular formula of 1 was established to be C₇₉H₁₃₁N₃O₂₀ by HRFABMS measurements. A 180 μL sample of 11n DMF-d₇ was prepared and ¹H,COSY, TOCSY, ROESY, and HSQC NMR data were obtained at 800 MHz using a 3mm room temperature probe. HMBC data, which were required for thestructural elucidation of 1, were acquired at 500 MHz using a 5 mm HCNinverse-detection Varian Chili-Probe in which the radio frequency coilsand preamplifier were cooled to approximately 25 K and 60 K,respectively. The NMR data (Table 1) established that poecillastrin A(1) was a new, highly functionalized macrolide, structurally related tothe chondropsins (Cantrell et al., J. Am. Chem. Soc. 2000, 122,8825-8829; Rashid et al., Tetrahedron Lett. 2001, 42, 1623-1626; Rashidet al., J. Nat. Prod. 2001, 64, 1341-1344).

The portion of 1 from C-5 to C-24 contained a chain of 20 contiguousprotonated carbons, and it was possible using the 800 MHz NMR data toassemble this structural fragment along with the associated methyl andoxygenated substituents. The section of 1 that encompassed C-25 to C-29was assigned by proton-proton spin system analysis and HMBC data. Thespin system that extended from C-30 through to C-68 was assigned in asimilar manner. No vicinal coupling was observed between the protons onC-41 and C-42, so ultimately the connection between C-41 and C-42 wasassigned, based on ROESY interactions observed between H-41 and H-67.The C-32 oxymethine proton had a downfield chemical shift (δ_(H) 5.26),which indicated that the oxygen at position 32 was esterified. This wasconfirmed by an HMBC correlation from H-32 to an ester carbonyl at δ_(C)172.1. COSY and HMBC correlations established that the ester carbonylwas part of an amino malic acid moiety that was attached to C-4 (δ_(C)166.6) via an amide bond. Thus, poecillastrin A (1) contains a33-membered macrocyclic ring that incorporates an amide bond between N-3and C-4, and an ester link between C-1 and the oxygen on C-32. An HMBCcorrelation from the N-43 proton (δ_(H) 7.38) to the C-44 carbonyl(δ_(C) 176.7) defined another amide linkage, while homonuclear protoncouplings and HMBC data allowed extension of the acyclic portion of 1out through the gem dimethyl substituents on C-75. The remainingstructural fragment from C-57 to C-65 was assembled using protoncoupling data to define the spin systems and HMBC correlations to bridgethe nonprotonated carbons. Attachment of this fragment through anα,β-unsaturated amide bond was established by HMBC correlations fromboth NH-56 (δ_(H) 7.70) and H-59 (δ_(H) 6.90) to C-57 (δ_(C) 164.8). Thegeometry of the olefin bonds in 1 were assigned as all E based onproton-proton couplings and ROESY interactions. Stereochemicalassignments for 1 were not attempted due to the large number ofasymmetric carbons (24) in the molecule and the small sample size.

TABLE 1 ¹H and ¹³C NMR Data for Poecillastrin A (1) in DMF-d₇ ^(a)position d_(C) mult d_(H) mult (J in Hz) HMBC position d_(C) mult d_(H)mult (J in Hz) HMBC 1 172.1 s 42 54.7 d 3.89 dd (9.0, 4.0) C-67 2 56.3 d5.10 dd (9.5, 2.2) C-34 43 7.38 d (9.0) C-44 3 8.21 d (9.5) C-4 44 176.7s 4 166.6 s 45 47.2 d 2.50 pent (7.0) C-44, C-46, C-47, C-69 5 124.0 d6.34 d (15.2) C-4, C-7 46 74.1 d 3.51 m C-44, C-69 6 141.3 d 7.12 dd(15.2, 11.0) C-4, C-5, C-7, C-8 47 33.5 t 1.51 m, 2H 7 129.8 d 6.26 mC-5, C-8, C-9 48 30.2 t 1.29 m, 2H 8 142.0 dt 6.08 d (15.0, 10.4) C-6,C-9 49 36.5 d 1.56 m 9 33.9 t 2.23 m, 2.31 m 50 83.5 d 3.57 m C-52, C-7110 32.8 t 2.19 m, 2H 51 136.1 s 11 134.5 d 5.82 dt (15.0, 8.0) 52 135.1d 5.47 bs C-50, C-54, C-71, C-72, C-73 12 131.2 d 6.25 m C-10, C-14 5340.8 s 13 134.5 d 6.36 dd (15.2, 11.0) C-11, C-15 54 81.7 d 3.51 m C-52,C-55, C-74 14 131.5 d 5.54 dd (15.2, 8.3) C-12 55 50.3 d 4.18 m 15 80.9d 3.98 m, C-13, C-16, OCH₃ 56 7.70 d (9.6) C-55, C-57 16 75.5 d 3.55 mC-15, C-18, C-20 57 164.8 s 17 34.4 t Hb 1.14 m, Ha 1.86 m 58 124.7 d6.16 d (15.2) C-57, C-60 18 26.0 d 1.85 m 59 146.8 d 6.90 d (15.2) C-57,C-58, C-60, C-61, C-79 19 41.5 t Hb 0.80 m, Ha 1.57 m 60 51.2 s 20 68.5d 3.55 m 61 215.3 s 21 43.6 t 1.29 m, 1.43 m 62 44.8 d 3.15 dq (9.6,7.0) C-61, C-63, C-80 22 66.1 d 4.18 m 63 77.7 d 3.52 m 23 41.6 d 1.45 m64 29.8 d 1.74 m C-65, C-81 24 80.1 d 3.82 d (9.6) C-22, C-23, C-26,C-37 65 14.2 q 0.82 d (7.0), 3H C-63, C-64, C-81 25 138.1 s 66 10.1 q0.94 d (7.0), 3H C-32, C-40, C-41 26 136.1 d 5.17 bd (8.0) C-24, C-37 6770.6 d 3.99 m 27 37.1 d 2.56 m 68 20.8 q 1.11 d (6.0), 3H C-42, C-67 2881.7 d 3.65 d (9.0) C-26, C-27, C-29, C-30 69 15.9 q 1.16 d (7.0), 3HC-44, C-45, C-46 29 138.9 s 70 15.6 q 0.90 d (7.0), 3H C-48, C-49, C-5030 122.0 d 5.27 m C-28, C-39 71 13.7 q 1.65 s, 3H C-50, C-51, C-52 3131.1 t 2.35 m, 2H C-29, C-30, C-32 72 25.4 q 1.10 s, 3H C-52, C-53, C-5432 75.3 d 5.26 m C-1, C-41, C-66 73 26.9 q 1.18 s, 3H C-52, C-53, C-5433 73.3 d 4.87 d (2.2) C-1 74 40.8 t 1.46 m, 1.54 m 34 171.7 s 75 25.3 d1.53 m 35 22.9 q 0.88 d (6.5), 3H C-17, C-18, C-19 76 24.5 q 0.84 d(7.0), 3H C-74, C-75, C-77 36 10.0 q 0.60 d (6.8), 3H C-22, C-23, C-2477 21.8 q 0.86 d (7.0), 3H C-74, C-75, C-76 37 11.4 q 1.60 s, 3H C-24,C-25, C-26 78 23.7 q 1.21 s, 3H C-59, C-60, C-61 38 17.8 q 0.76 d (6.8),3H C-26, C-27, C-28 79 23.9 q 1.27 s, 3H C-59, C-60, C-61, C-78 39 12.3q 1.59 s, 3H C-28, C-29, C-30 80 15.7 q 0.83 d (7.0), 3H C-61, C-62,C-63 40 40.3 d 1.84 m 81 20.7 q 0.92 d (7.0), 3H C-63, C-64, C-65 4174.3 d 3.66 m OCH₃ 56.4 q 3.23 s, 3H C-15 ^(a13)C assignments were madeusing HSQC and HMBC data, and multiplicities inferred using amultiplicity-edited HSQC pulse sequence.

Example 3

This example illustrates the general procedure for obtaining theactivity profile of compounds of the present invention using the NCI 60cell-line screen.

An extract from Poecillastra species was tested in the NCI 60 cell-linescreen as described in detail in Boyd and Paull, Drug Dev. Res., 34,91-109 (1995); and Monks et al., J. Natl. Cancer Inst., 83, 757-766(1991). Briefly, a stock solution of the extract was prepared initiallyin dimethylsulfoxide at 400× the desired final highest testconcentrations and stored at −70° C. until use. The final highest testconcentrations studied in this example varied between 10⁻⁵ and 10⁻⁸molar. At the time of screening, an aliquot of the thawed stock wasdiluted with complete medium containing 50 μg/ml gentamycin to give aconcentration of 2× the desired final highest test concentration. Fouradditional 10-fold serial dilutions were then made to provide a total offive concentrations, spanning a 4-log₁₀ concentration range. One hundredμl aliquots of these intermediate dilutions were immediately added tothe appropriate microtitre wells, each already containing theappropriate numbers and types of cells in 100 μl of culture medium,resulting in the desired five final concentrations.

The 60 cell lines used, and the respective inoculation densities, wereas described in Boyd and Paull, supra, and Monks et al., supra.Following the compound additions, the plates were incubated for 48 h at37° C. under a 5% CO₂/air atmosphere and 100% humidity. Then, adherentcells (all lines except the leukemia) were fixed in situ by gentleaddition of cold trichloroacetic acid (50 μl of 50% w/v) and incubatedfor 60 min at 4° C. Supernatants were discarded, and plates were washedfive times with deionized water and air-dried. Sulforhodamine B solution(SRB; 100 μl at 0.4% w/v in 1% acetic acid) was added to each plate,followed by further incubation for 10 min at room temperature. Excessunbound dye was then removed by washing five times with 1% acetic acid,followed by air-drying. The bound stain in each well was solubilized byaddition of 100 μl of 10 mM unbuffered Tris base; this was followed by adetermination of optical densities (515 nm) on an automated platereader. For suspension cell cultures (the leukemias), the method was thesame except that, at the end of the drug incubation period, the settledcells were fixed in situ to the bottoms of the microtitre wells bygentle addition of 50 μl of 80% trichloroacetic acid. Appropriatecontrol wells were included in the test plate format (Monks et al., J.Natl. Cancer Inst., 83, 757-766 (1991)) to allow subtraction ofbackground optical densities, drug-blank corrections, and adetermination of cell densities at time 0 (the time at which compoundsare added).

The testing of extract of Poecillastra species in the NCI 60 cell-linescreen gave the characteristic GI₅₀-based and TGI-based mean-graph“fingerprints” in the NCI 60-cell screen exemplified in FIGS. 1A, 1B and1C. The following averaged, individual negative log₁₀ GI₅₀ values, shownalong with the respective subpanel and cell-line identifiers, wererecorded for the extract of Poecillastra species (of which poecillastrinA is a major component): (Leukemia) CCRF-CEM (<0.40), HL-60-TB (<0.40),K-562 (<0.40), MOLT-4 (<0.40), RPMI-8226 (1.89), SR (<0.40); (Non-SmallCell Lung) A549/ATCC (<0.40), EKVX (1.34), HOP-18 (1.30), HOP-62(<0.40), NCI-H226 (<0.40), NCI-H23 (1.87), NCI-H322M (1.87), NCI-H460(<0.40), NCI-H522 (0.73), LXFL529 (<0.40); (Small Cell Lung) DMS114(0.97), DMS273 (<0.40); (Colon) COL0205 (<0.40), DLD-1 (1.11), HCC-2998(<0.40), HCT-116 (<0.40), HCT-15 (<0.40), HT29 (<0.40), KM12 (<0.40),KM20L2 (<0.40), SW-620 (<0.40); (CNS) SF-268 (1.55), SF-295 (<0.40),SF-539 (1.42), SNB-19 (2.01), SNB-75 (1.44), SNB-78 (1.55), U251 (1.63);(Melanoma) LOX-IMVI (<0.40), MALME-3M (<0.40), M14 (<0.40), SK-MEL-2(<0.40), SK-MEL-28 (<0.40), SK-MEL-5 (<0.40), UACC-257 (<0.40), UACC-62(<0.40); (Ovary) IGROV1 (1.45), OVCAR-3 (1.11), OVCAR-4 (<0.40), OVCAR-5(1.97), OVCAR-8 (<0.40), SK-OV-3 (1.51); (Renal) 786-0 (0.95), A498(1.02), ACHN (1.42), CAKI-1 (0.97), SN-12C (1.02), TK-10 (1.45), UO-31(<0.40).

GI₅₀ and TGI-COMPARE analyses of the full data set obtained from thescreening of the extract of Poecillastra species revealed that thecompound gave a striking pattern of differential cytotoxicity in the NCI60 cell-line screen that is characteristic of compounds of the presentinvention (e.g., Pearson correlation coefficients greater than or equalto 0.7-0.8) but unlike that of any known conventional anticancer drugclass.

Example 4

This example demonstrates the vacuolar-type (H+)-ATPase inhibitoryactivity of an extract of Poecillastra species.

This method employs the NCI 60 cell-line in vitro screen to obtain amean-graph “fingerprint” of a desired mechanistic prototype compound,then using a computer-based search algorithm called COMPARE, to search adatabase of mean-graph “fingerprints” of structurally unrelatedcompounds to thereby identify compounds with fingerprints very similar,if not indistinguishable, from that of the selected prototype (or“seed”). The degree of similarity is determined by calculation of aCOMPARE correlation coefficient, which can vary from a lowest value ofzero (which indicates no correlation), to a highest value of one (whichindicates a perfect correlation). A high COMPARE correlation (i.e.,indicating a high degree of similarity) between the mean-graph“fingerprints” of different compounds indicates that the compounds acton the same or similar molecular target and, therefore, shareessentially the same or similar mechanism of biological activity. Inpractical terms, a COMPARE correlation coefficient of about 0.9 orhigher indicates that, within the limits of experimental error of thescreening process, the mean-graph “fingerprints” of the comparedcompounds are essentially identical or indistinguishable and, therefore,that the compounds act on the same molecular target. For pertinentbackground on the NCI 60 cell-line screen and the method andapplications of COMPARE, see Boyd, In: Current Therapy in Oncology(Niederhuber, J. E., ed) Philadelphia: B. C. Decker, 1993, pp. 11-22;Boyd and Paull, supra; Paull et al., In: Cancer Chemotherapeutic Agents,Washington, D.C.: Am. Chem. Soc. Books, 1995, pp. 11-45.

Potent known vacuolar-type (H+)-ATPase inhibitors, (see, e.g., Boyd, PCTInternational Patent Application No. PCT/US00/05582), lobatamide A,bafilomycin A₁, concanamycin A and salicylihalamide A were selected foruse as comparative examples. For pertinent background on concanamycinsand bafilomycins, see Bowman et al., Proc. Natl. Acad. Sci. USA, 85,7972-7976 (1988); Dröse et al., Biochemistry, 32, 3902-3906 (1993);Dröse and Altendorf, J. Exp. Biol., 200, 1-8 (1997). For pertinentbackground on lobatamide A and salicylihalamide A, see Boyd, PCTInternational Patent Application No. PCT/US00/05582.

In the present example, authentic, well characterized and documentedreference samples of concanamycin A and bafilomycin A₁ were obtainedfrom a commercial supplier (Kamiya Biochemical Company, Tukwila, Wash.).Salicylihalamide A and lobatamide A were obtained as described by Boyd,PCT International Patent Application No. PCT/US00/05582.

As appropriate for this demonstration, the TGI mean-graph, derived fromthe contemporaneous testing of an extract of Poecillastra species, wasused as the “seed” to search against the TGI mean-graphs and LC₅₀mean-graphs contained in the aforementioned database, and as the basisfor calculation of the COMPARE coefficients.

Table 2 summarizes the TGI-COMPARE and LC₅₀-COMPARE correlationcoefficients from the testing of lobatamide A, concanamycin A,bafilomycin A₁, salicylihalamide A and an extract of Poecillastraspecies in the NCI 60 cell-line screen. The mean-panel GI₅₀ values arealso shown in Table 2.

TABLE 2 TGI-COMPARE Correlation Compound Coefficient LC₅₀'s Lobatamide A0.50 0.66 Concanamycin A 0.43 0.47 Bafilomycin A₁ 0.54 0.65Salicylihalamide A 0.61 0.60 Extract of Poecillastra 1.00 1.00 species

As shown in Table 2, this analysis correctly identified compounds which,although structurally distinct from the seed, nonetheless shared thesame molecular target (i.e., in this instance, vacuolar-type(H+)-ATPase). Compounds of the present invention can exhibit a range ofrelative absolute potencies against vacuolar-type (H+)-ATPase.

Example 5

This example describes the cytoxicity of poecillastrin A (1), isolatedin Example 1. The cytotoxicity assay, details of which have beendescribed previously (Bokesch et al., J. Nat. Prod. 1999, 62, 633-635),utilized melanoma (LOX), breast (A-549), ovarian (OVCAR-3), andnon-small cell lung (SNB-19) human tumor cell lines and IC-2^(WT) andIC-2^(V814) murine mast cell lines (Hashimoto et al., Am. J. Pathol.1996, 148, 189-200).

The in vitro 10 cell-line bioassay was a two day bioassay. Cells weregrown in RPMI-1640 (Rosweli Park Memorial Institute) withoutL-glutamine, supplemented with 10% fetal bovine serum, 5.0 mL of a 200mM glutamine stock, and 0.5 mL of gentamicin and plated out in T-162 cm²flasks. Once the cells were confluent, they were harvested and plated in96-well microtiter flat-bottom plates at a seeding density of 50-10,000cells per well, to yield optical density readings in the range of 1-2.0,and incubated for 1 h in a 37° C., 5% CO₂ incubator. After the 1 hincubation, the cells were then introduced to the sample ofpoecillastrin A, via a Beckman Biomek Workstation-1000. The Biomek-1000performed seven serial dilutions in a 96-well round-bottom plate andthen transferred aliquots of 100 μL to the assay plate. The plate wasthen returned to the incubator for 24 h. After the two day incubation,the cells were exposed to tetrazolium salt,2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide(XTT), for a 4 h incubation in a 37° C. incubator, where viable cellsreduced the tetrazolium salt to a colored formazan product. Once theincubation was complete, the plates were read in a dual wavelength modeat 450 nm, with a 650 nm reference, using a SpectraMAX 250 (MolecularDevices) plate reader. Resulting data for each cell line are found inTable 3.

TABLE 3 Tumor Type Cell Line IC₅₀ (μM) Melanoma LOX 0.014 OvarianOVCAR-3 0.028 Breast A549 >10 Non-small cell lung SNB-19 >10 Murine mastcell IC-2 WT 0.206 Murine mast cell IC-2 V814 0.546

The data confirm that poecillastrin A is both a potent and differentialcytotoxin, similar to the chondropsins.

Example 6

This example demonstrates the vacuolar-type (H+)-ATPase inhibitoryactivity of poecillastrin A using a vacuolar-type (H+)-ATPase inhibitionassay.

Poecillastrin A was tested for its inhibitory activity againstvacuolar-type (H+)-ATPases from bovine chromaffin granule membranes(“Bovine CGM V-ATPase”) and from vacuolar membranes of N. crassa (“Nc VMV-ATPase”). For Nc VM V-ATPase, strain 74A of N. crassa was used. Thestrain was maintained on Vogel's medium N (a minimal medium saltsolution at pH 5.8) supplemented with 2% sucrose. For membraneisolations, cells were grown approximately 14 hours at 25° C. in 4liters of Vogel's medium inoculated with 10⁶ conidia/ml (asexual spores)and aerated vigorously.

Chromaffin granule membranes were prepared from bovine adrenal glands,obtained fresh from a local abattoir, as described by Nelson et al.,Methods Enzymol., 157, 619-633 (1988). The membranes were stored inaliquots at −70° C. Vacuolar membranes were prepared from N. crassa asdescribed by Bowman et al., Biomembranes (Packer, L., and Fleischer, S.,eds), pp. 861-872, Academic Press San Diego (1997), and modified Bowmanet al., J. Biol. Chem., 272, 14776-14786 (1997).

Protein and ATPase activities were assayed as described by Bowman etal., J. Biol. Chem., 272, 14776-14786 (1997), except that assays weredone at 37° C. Poecillastrin A was added to assay mixtures from 5 or 10mM stock solutions in dimethyl sulfoxide. When comparing the effects ofthe inhibitor on different membranes, the reactions were run at the sametime in the same assay mix. Vacuolar-type (H+)-ATPase activities weremeasured at 37° C. with 2 μg of vacuolar membrane protein and variousconcentrations of poecillastrin A. Specific activities in the absence ofinhibitor were 0.18 μmol/min/mg of protein for the Bovine CGM V-ATPase,and 5.0 mmol/min/mg of protein for the Nc VM V-ATPase. Each value wasbased on the average of three independent titrations. The results of theassay are shown in Table 4.

TABLE 4 K_(i) for Bovine CGM K_(i) for Nc VM Compound V-ATPase (μM)V-ATPase (μM) Poecillastrin A 8.0 0.40

The foregoing data demonstrate that poecillastrin A is an effectiveinhibitor of vacuolar-type (H+)-ATPase, exhibiting K_(i) values in themicromolar to sub-micromolar range.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-15. (canceled)
 16. A method of preventing or treating a patient forcancer, which method comprises administering to the patient ananticancer effective amount of at least one compound of the formula:

wherein: R¹ is H, a straight-chain or branched C₁₋₃₀ saturated alkyl, astraight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹ isunsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(1a), CO₂R^(1a), and OC(O)R^(1a), wherein R^(1a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; R²-R⁸ are the same or different andeach is R¹⁰, C(O)R¹⁰, SO₃R¹⁰, or SO₂R¹⁰, wherein R¹⁰ is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof, wherein R¹⁰ is unsubstituted orsubstituted with one or more substituents, which are the same ordifferent, selected from the group consisting of a halogen, an oxo,OR^(10a), CO₂R^(10a) and OC(O)R^(10a), wherein R^(10a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; and R⁹ is a substituent of theformula:

wherein the R^(9a) substituents are the same or different and each isR¹¹, C(O)R¹¹, or SO₂R¹¹, wherein R¹¹ is H, a straight-chain or branchedC₁₋₃₀ saturated alkyl, a straight-chain or branched C₂₋₃₀ unsaturatedalkyl, or an aryl comprising 6-10 carbon atoms in the ring skeletonthereof, wherein R¹¹ is unsubstituted or substituted with one or moresubstituents, which are the same or different, selected from the groupconsisting of a halogen, an oxo, OR^(11a), CO₂R^(11a) and OC(O)R^(11a),wherein R^(11a) is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof; whereinR^(1a), R^(10a) and R^(11a) are unsubstituted or substituted with one ormore substituents selected from the group consisting of a halogen, anoxo, and a hydroxyl; or a pharmaceutically acceptable salt thereof,whereupon the patient is treated for cancer.
 17. The method of claim 16,wherein the method further comprises administering an anticancereffective amount of at least one additional compound other than acompound of formula (I), which is an anti-cancer compound.
 18. Themethod of claim 17, wherein the at least one additional compound is asalicylihalamide.
 19. The method of claim 16, wherein the cancer isselected from the group consisting of human leukemias, lymphomas,melanomas and solid tumors.
 20. The method of claim 19, wherein thesolid tumor is selected from the group consisting of lung cancer, coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer, head and neck cancer, testicular cancer, germ-line cancers,endocrine tumors, uterine cancer, breast cancer, sarcomas, gastriccancer, hepatic cancer, esophageal cancer and pancreatic cancer.
 21. Themethod of claim 16, wherein the cancer is selected from the groupconsisting of colon cancer, melanoma, breast cancer, ovarian cancer andnon-small lung cancer.
 22. A method of inhibiting vacuolar-type(H+)-ATPase in a patient in need thereof, the method comprisingadministering to the patient a vacuolar-type (H+)-ATPase-inhibitingeffective amount of at least one compound of the formula:

wherein: R¹ is H, a straight-chain or branched C₁₋₃₀ saturated alkyl, astraight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof, wherein R¹ isunsubstituted or substituted with one or more substituents, which arethe same or different, selected from the group consisting of a halogen,an oxo, OR^(1a), CO₂R^(1a), and OC(O)R^(1a), wherein R^(1a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; R²-R⁸ are the same or different andeach is R¹⁰, C(O)R¹⁰, SO₃R¹⁰, or SO₂R¹⁰, wherein R¹⁰ is G, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof, wherein R¹⁰ is unsubstituted orsubstituted with one or more substituents, which are the same ordifferent, selected from the group consisting of a halogen, an oxo,OR^(10a), CO₂R^(10a) and OC(O)R^(11a), wherein R^(10a) is H, astraight-chain or branched C₁₋₃₀ saturated alkyl, a straight-chain orbranched C₂₋₃₀ unsaturated alkyl, or an aryl comprising 6-10 carbonatoms in the ring skeleton thereof; and R⁹ is a substituent of theformula:

wherein the R^(9a) substituents are the same or different and each isR¹¹, C(O)R¹¹, or SO₂R¹¹, wherein R¹¹ is H, a straight-chain or branchedC₁₋₃₀ saturated alkyl, a straight-chain or branched C₂₋₃₀ unsaturatedalkyl, or an aryl comprising 6-10 carbon atoms in the ring skeletonthereof, wherein R¹¹ is unsubstituted or substituted with one or moresubstituents, which are the same or different, selected from the groupconsisting of a halogen, an oxo, OR^(11a), CO₂R^(11a) and OC(O)R^(11a),wherein R^(11a) is H, a straight-chain or branched C₁₋₃₀ saturatedalkyl, a straight-chain or branched C₂₋₃₀ unsaturated alkyl, or an arylcomprising 6-10 carbon atoms in the ring skeleton thereof; whereinR^(1a), R^(10a) and R^(11a) are unsubstituted or substituted with one ormore substituents selected from the group consisting of a halogen, anoxo, and a hydroxyl; or a pharmaceutically acceptable salt thereof,whereupon vacuolar-type (H+)-ATPase in the patient is inhibited.
 23. Themethod of claim 22, wherein: R¹ is H or a straight-chain or branchedC₁₋₃₀ saturated alkyl; R²-R⁸ are the same or different and each is R¹⁰,wherein R¹⁰ is H or a straight-chain or branched C₁₋₃₀ saturated alkyl;and the R^(9a) substituents are the same or different and each is R¹¹,wherein R¹¹ is H or a straight-chain or branched C₁₋₃₀ saturated alkyl;or a pharmaceutically acceptable salt thereof, whereupon vacuolar-type(H+)-ATPase in the patient is inhibited.
 24. The method of claim 23,wherein: R¹ is H or a straight-chain or branched C₁₋₆ saturated alkyl;R²-R⁸ are the same or different and each is R¹⁰, wherein R¹⁰ is H or astraight-chain or branched C₁₋₆ saturated alkyl; and the R^(9a)substituents are the same or different and each is R¹¹, wherein R¹¹ is Hor a straight-chain or branched C₁₋₆ saturated alkyl; or apharmaceutically acceptable salt thereof, whereupon vacuolar-type(H+)-ATPase in the patient is inhibited.
 25. The method of claim 22,wherein: the compound is a compound of the formula:

or a pharmaceutically acceptable salt thereof, whereupon vacuolar-type(H+)-ATPase in the patient is inhibited.
 26. The method of claim 25,wherein: the compound is a compound of the formula:

or a pharmaceutically acceptable salt thereof, whereupon vacuolar-type(H+)-ATPase in the patient is inhibited.
 27. The method of claim 22,wherein the patient is affected by a condition treatable or preventableby inhibition of vacuolar-type (H+)-ATPase.
 28. The method of claim 27,wherein the condition is selected from the group consisting ofosteoporosis, Alzheimer's disease, glaucoma, fertility, abnormal urinaryacidification, abnormal secretion of degradative enzymes, fungalinfection and cancer.
 29. The method of claim 27, wherein thevacuolar-type (H+)-ATPase inhibiting-effective amount is effective toinhibit one or more conditions selected from the group consisting ofintra-organellar acidification of intracellular organelles, urinaryacidification, bone resorption, fertility, drug-resistance of tumorcells, tumor cell proliferation, cellular invasiveness, angiogenesis,and metastasis.
 30. The method of claim 27, wherein the method furthercomprises administering a vacuolar-type (H+)-ATPase inhibiting-effectiveamount of at least one additional compound other than a compound offormula (I), which inhibits vacuolar-type (H+)-ATPase.
 31. The method ofclaim 30, wherein the at least one additional compound is asalicylihalamide.